THE JOURNAL OF COMPARATIVE NEUROLOGY 305337-347 (1991)
Afferent and Efferent Connections of the Olfactory Bulbs in the Lizard Podarcis hispanica FERNANDO MARTINEZ-GARCIA, FRANCISCO E. OLUCHA, VICENT TERUEL, MARIA J. LORENTE, AND WALTER K. SCHWERDTFEGER Universitat de Valencia, Facultat de Cikncies Biolbgiques, Departament de Biologia Animal. Unitat de Morfologia Microscbpica. Spain (F.M.-G., F.E.O., V.T., M.J.L.),and Max-Planck Institut fur Hirnforschung, Frankfurt am Main, Germany (W.K.S.)
ABSTRACT The connections of the olfactory bulbs of Podarcis hispanica were studied by tract-tracing of injected horseradish peroxidase. Restricted injections into the main olfactory bulb (MOB) resulted in bilateral terminallike labeling in the medial part of the anterior olfactory nucleus (AON) and in the rostral septum, lateral cortex, nucleus of the lateral olfactory tract, and ventrolateral amygdaloid nucleus. Bilateral retrograde labeling was found in the rostral lateral cortex and in the medial and dorsolateral AON. Ipsilaterally the dorsal cortex, nucleus of the diagonal band, lateral preoptic area, and dorsolateral amygdala showed labeled cell bodies. Retrogradely labeled cells were also found in the midbrain raphe nucleus. Results from injections into the rostral lateral cortex and lateral olfactory tract indicate that the mitral cells are the origin of the centripetal projections of the MOB. Injections in the accessory olfactory bulb (AOB) produced ipsilateral terminallike labeling of the ventral AON, bed nucleus of the accessory olfactory tract, central and ventromedial amygdaloid nuclei, medial part of the bed nucleus of the stria terminalis, and nucleus sphericus. Retrograde labeling of neurons was observed ipsilaterally in the bed nucleus of the accessory olfactory tract and stria terminalis, in the central amygdaloid nucleus, dorsal cortex, and nucleus of the diagonal band. Bilateral labeling of somata was found in the ventral AON, the nucleus sphericus (hilus), and in the mesencephalic raphe nucleus and locus coeruleus. Injections into the dorsal amygdala showed that the mitral neurons are the cells of origin of the AOB centripetal projections. Reciprocal connections are present between AOB and MOB. To our knowledge, this is the first study to address the afferent connections of the olfactory bulbs in a reptile. On the basis of the available data, a discussion is provided of the similarities and differences between the reptilian and mammalian olfactory systems, as well as of the possible functional role of the main olfactory connections in reptiles. Key words: comparative neuroanatomy,HRP, reptile, amygdala, vomeronasal system, olfactory system
In amphibians, squamate reptiles (lizards and snakes), and the majority of mammals, chemical signals enter the telencephalon in two different ways: from the nasal epithelium via the main olfactory bulbs (MOB), and from the vomeronasal organ via the accessory olfactory bulbs (AOB). Experimental studies on the behavior of reptiles (for review, see Burghart, ’70, Halpern, ’87) indicate that this kind of information (chemosensory signals from both the olfactory and vomeronasal systems) is essential for these animals in several biologically significant activities, such as sexual behavior and foraging. Moreover, the olfactory structures of the brain, especially those of the vomeronasal system, are especially well developed in snakes and terrestrial lizards (Bellairs, ’69; Gabe and Saint Girons, ’76).
o 1991 WILEY-LISS, INC.
Other anatomical features accompany the high development of the accessory olfactory system in those reptiles, namely, the presence of a forked tongue useful to introduce “odorant” particles into the Jacobson’s organ. In spite of the importance of olfaction for the biology of reptiles, anatomical investigations on fiber connections of their olfactory bulbs have so far been restricted to the study of their centripetal projections, using anterograde degeneration techniques (Gamble, ’52; Heimer, ’69; Halpern, ’76; Accepted October 31,1990 Address reprint requests to Fernando Martinez-Garcia, Dept. Biologia Animal, Unitat de Morfologia Microscopica. Fac. C. Biologiques, Univ. Valencia. C. Dr. Moliner, 50. E-46100 Burjassot, Spain.
F. MARTINEZ-GARCIA E T AL.
338
Ulinski and Peterson, ’81; Lohman et al., ’88),anterograde transport of tritiated aminoacids (Reiner and Karten, ’851, and Phaseolus uulgaris leucoagglutinin (Lohman et al., ’88). The knowledge of the olfactory bulb afferents should be equally essential for understanding the structure and the physiology of the vomeronasal and olfactory systems. Hence, in the present study, anterograde and retrograde transport of horseradish peroxidase (HRP) were used in order to: (1)map the origin of afferent projections to the bulbs, and (2) check the efferents of the olfactory bulbs with a tracing technique different from those that have been previously applied.
MATERIAL AND METHODS For this study 35 adult specimens of the lizard Podarcis hispanica (45-60 mm snout-vent length) were used. Under ether anaesthesia, 10-20 nl of 20% HRP (Sigma, type VI) in 2% dimethyl sulfoxide or 10% Saponin (Merck, Darmstadt, F.R.G.) were injected in the main olfactory bulb (MOB) of 16 specimens, and in the accessory olfactory bulb (AOB) of 11lizards. Additional HRP injections were performed in the rostral lateral cortex (4 specimens) and in the amygdala (dorsolateral amygdaloid nucleus and nucleus sphericus; 4 lizards). After 7-9 days survival a t room temperature (18-2loC), animals were anaesthetized again and were transcardially perfused with saline followed by cold (4°C) fixative containing 1.25% glutaraldehyde and 1% paraformaldehyde in 0.1 M phosphate buffer, pH 7.2. Brains were immersed in the same fixative for 4 hours and rinsed overnight at 4°C in 30% phosphate-buffered sucrose. Fifty-pm-thick frontal sections of the brains were obtained using a freezing microtome. Sections were processed according to the tetramethyl benzidine-ammonium heptamolybdate method for histochemical detection of peroxidase activity (Olucha et al., ’85), mounted in chromalumcoated slides, and counterstained with neutral red.
RESULTS Prior to reporting the results of HRP injections, we describe briefly the organization of relevant regions of the telencephalic hemispheres in Podarcis. The terminology
employed for this and for the hodological study allows us to compare our results for Podarcis with those of other authors for different reptilian species.
Terminology The rostral tip of the telencephalic hemisphere plus the olfactory peduncle are known as the “retrobulbar formation” (RBF). Within the RBF two superficial structures showing a laminar arrangement can be distinguished: on the one hand, the lateral cortex, that is, the rostralmost cortical area and, on the other hand, the ventral olfactory tubercle (OT) (Fig. lA,B). The “core” of the RBF is occupied by a complex structure that we have called, according to the classical nomenclature, anterior olfactory nucleus (AON). Three different subnuclei are distinguished within the AON with respect to neuronal packing and topographical position (Fig. 1A,B). The laterodorsal AON (AON1) is formed by a group of scattered neurons just below the lateral cortex. It displays a large rostrocaudal extent, being evident between the caudal margin of the olfactory bulbs and the rostral margin of the dorsal cortex and pallial thickening. These features resemble those of the region called “polymorphic layer of the dorsal cortex” by Halpern (’80). The dorsomedial AON (AONm) (Fig. 1B) displays a high density of neuronal packing. It coincides in location and neuronal density with the dorsomedial retrobulbar formation of Halpern (’80). Moreover, it is caudally bounded by the medial cortex showing a diffuse trilaminar organization; for this reason a similar region in the rostral telencephalon of Dipsosaurus dorsalis was called by Ulinski and Peterson (’81) “rostral rudiment of the medial cortex.” Finally, the ventral AON (AONv) (Fig. 1A) is made up of a compact group of juxtaependymal neurons, caudally bounded by the rostral ADVR. It roughly resembles the retrobulbar formation of Ulinski and Peterson (’81). The olfactory tubercle is rostrally adjacent to the nucleus of the diagonal band (DBN) (Fig. 1C). The limit between both structures is rather diffuse in a Nissl preparation, but it is clear in terms of fiber connectivity, e.g., only the DBN projects to the cerebral cortex (Bruce and Butler, ’84; Martinez-Garcia et al., ’86). The DBN reaches the caudal tip of the telencephalon, up to diencephalic levels (caudally
Abbreviations
ac ACC
ADVR AOB aot AON AONl AONm AONv BAOT BNM BST CAN D DEN DLAN
DM gl HRP ipl iot
anterior commisure nucleus accumbens anterior dorsal ventricular ridge accessory olfactory bulb accessory olfactory tract anterior olfactory nucleus laterodorsal anterior olfactory nucleus dorsomedial anterior olfactory nucleus ventral anterior olfactory nucleus bed nucleus of the accessory olfactory tract bed nucleus of the medial forebrain bundle bed nucleus of the stria terminalis central amygdaloid nucleus dorsal cortex diagonal band nucleus dorsolateral amygdaloid nucleus dorsomedial cortex granular layer horseradish peroxidase inner plexiforrn layer intermediate olfactory tract
L LCO Irn lot LPA
M mcl MOB Mot mot Nlot NS OT
R RBF S srn STR
VL VM
lateral cortex locus coeruleus lateral forebrain bundle lateral olfactory tract lateral preoptic area medial cortex mitral cell layer main olfactory bulb main olfactory tract medial olfactory tract nucleus of the lateral olfactory tract nucleus sphericus olfactory tubercle midbrain raphe nucleus retrobulbar formation septum stria rnedullaris striatum ventrolateral amygdaloid nucleus ventromedial amygdaloid nucleus
LIZARD OLFACTORY BULB CONNECTIONS
Fig. 1. Nissl-stained transverse sections of the right telencephalic hemisphere of Podarcis hispanica showing the cytoarchitecture of the retrobulbar formation (A and B) amygdaloid formation (D and E)and other olfactory structures. Bar = 250 pm.
339
340
F. MARTINEZ-GARCIA ET AL.
Contralateral intermediate olfactory tract (iot). Fiber labeling appeared throughout the contralateral iot. At rostral levels this tract ascended in a midsagittal position through the rostral septum, where labeled fibers may perform synaptic contacts. In connection with this tract, labeled puncta and somata were observed in the AONm (Fig. 2C). The terminal-like labeling was restricted to the outer half of layer 1. Zpsilateral intermediate olfactory tract. Apparent labeling was present within the ipsilateral iot throughout its rostrocaudal extent. At rostral levels it occupied the outer layer of the olfactory tubercle where not only fiber labeling occurred but also intense terminallike labeling, suggesting the presence of synaptic contacts. Some smooth labeled fibers of the iot left the tract and entered the medial forebrain bundle at the level of either the nucleus accumbens or the nucleus of the medial forebrain bundle (Fig. 2D). The ipsilateral diagonal band nucleus showed retrograde labeling (see Fig. 4C) of neurons located mainly in the “horizontal limb.” They were usually fusiform with their main axis parallel to the pial surface. However, some stellate labeled neurons were also observed. This retrograde labeling continued beyond the caudal boundary of the DBN; several labeled neurons were found in the hypothalamic Injections of HRP into the main olfactory bulb lateral preoptic area (Fig. 2E), which sent their axons directly to the stria medullaris. After injections into the MOB, fiber labeling appeared Zpsilateral medial olfactory tract (mot). The mot takes bilaterally in the iot and lot, and in the ipsilateral mot (Fig. a midsagittal course giving rise to terminal fields in the 2). Contralateral connections were seen to course through rostral septum and in the AONm. Anterograde labeling the habenular commissure-striae medullaris system. In the showed the same laminar distribution as the contralateral following section we describe in detail the anterograde and side. Retrogradely labeled neurons were also observed in retrograde labeling found in connection with each labeled this part of the AON (Fig. 2 0 . tract. Accessory olfactory bulb. The ipsilateral accessory olfacContralateral lateral olfactory tract {lot). In the con- tory bulb showed terminallike labeling occupying the mitral tralateral hemisphere, the lateral cortex showed labeling of cell layer and part of the granular layer. Some retrogradely terminallike puncta and cell bodies (Fig. 2C). The terminal- labeled cells were observed in the mitral layer, indicating like labeling occupied the outer half of layer 1 (outer reciprocal connections between the MOB and AOB (Fig. 2B; plexiform layer) throughout its rostrocaudal axis (see Fig. see also Fig. 4D). 4A); a few retrogradely labeled somata were found in the Mesencephalon. At mesencephalic levels a few labeled lateral cortex at retrobulbar levels. Some neurons were also cells were found in the midbrain raphe nucleus (Fig. 2G). labeled in the AONl (Fig. 2C), whose axons were not detected. Since the nearest labeled tract to these neurons Injections of HRP into the accessory was the lot, this tract is likely to contain their axons. olfactory bulb Terminallike labeling was also observed in the so-called nucleus of the lot, in the lateral margin of the telencephalic After injections of HRP into the accessory olfactory bulb hemisphere. However, since this nucleus is immersed in the (AOB), intense fiber labeling was observed in the accessory lot, it is difficult to decide whether it actually corresponds to olfactory tract (aot). Some labeled axons were observed terminal labeling or to transversally cut fibers. Structures bilaterally in the intermediate olfactory tract (Fig. 3C,D,E) resembling boutons en passant were observed all along the and stria medullaris (Fig. 3F), as well as in the anterior tract and were especially frequent at the level of the (Fig. 3E) and habenular commissures. ventrolateral amygdala (see Fig. 4B). Zpsilateral accessory olfactory tract (aot). Reciprocal Zpsilateral lateral olfactory tract. The pattern of la- connections were observed between the AOB and the beled structures in connection with the ipsilateral lot was AONv, where retrograde and terminallike labeling was similar to that of the contralateral hemisphere, except for observed (Fig. 3 0 . Some labeled somata appeared in the the higher intensity of anterograde and fiber labeling and dorsal cortex, with the same distribution as seen after MOB the higher number of retrogradely labeled neurons. How- injections (Fig. 3D,E). ever, two main differences were noticed, namely retrograde The main target for the AOB centripetal connections is labeling of neurons in the dorsolateral amygdaloid nucleus the amygdaloid formation. At rostral levels, both retro(Fig. 2E), and in the cell layer of the caudal dorsal cortex grade and terminallike labeling was observed in the bed (Fig. 2D,E). At commissural levels, the medial and lateral nucleus of the accessory olfactory tract (Fig. 3C; belonging thirds of the dorsal cortex showed labeling, whereas the to the perifascicular complex of Ulinski and Peterson, ’81) intermediate portion remained unlabeled. The unlabeled and in the central amygdaloid nucleus. Caudally, a profuse intermediate portion decreased succesively at caudal levels. terminal field was found in the hilus of the nucleus Hence, at the caudal edge of the cortex, the whole dorsal sphericus, bordered by a narrow unlabeled band just below cortex showed labeled somata. the mural layer (Fig. 3E,F). This anterograde labeling
and ventrally to the anterior commissure), where it gradually looses its laminar organization and merges with the medial and lateral preoptic areas. The caudal edge of the ventrolateral telencephalon has been termed “archistriaturn” (e.g., Northcutt, ’67) and “basal dorsal ventricular ridge” (Ulinski, ’83). We prefer the classical terminology, or “amygdala” (Curwen, ’39). In Podarcis the most spectacular nucleus of the amygdala is the nucleus sphericus (Fig. 1E). A series of more rostrally located nuclei have been termed according to their topographical position: dorsolateral, ventrolateral, ventromedial, and central nuclei of the amygdala (Fig. 1D). At the levels of the latter nuclei, the ventral sulcus of the telencephalic ventricle is surrounded by a group of neurons crossed by the fibers of the stria terminalis. We therefore call it “bed nucleus of the stria terminalis” (Fig. 1D). Finally, our nomenclature of the olfactory tracts is based on Ulinski and Peterson (’81). We consider three different tracts related with the main accessory bulb (MOB); the lateral olfactory tract (lot),the medial olfactory tract (mot), and the intermediate olfactory tract (iot). The accessory olfactory tract (aot) connects the accessory olfactory bulb (AOB)with the telencephalic hemisphere.
LIZ ARDOLFACTORYBULBCONNECTIONS
341
iot Fig. 2. Schematic drawing of seven transverse sections of the brain of Podarcis showing the retrograde (open circles), fiber (lines)and terminallike labeling (dots) resulting from a restricted HRP injection (A) into the MOB.
extended into rostrally adjacent nuclei such as the ventromedial amygdaloid nucleus and the lateral part of the bed nucleus of the stria terminalis (Fig. 3F). Moreover, labeling of neuronal cell bodies was observed in these amygdaloid structures; in the nucleus sphericus labeled somata were
mainly located in the hilus but also in the mural layer (Fig. 4E). Finally, some retrogradely labeled hilar cells in the contralateral nucleus sphericus were seen to send their axons via the anterior commissure to the ipsilateral aot (Fig. 3E).
F. MARTINEZ-GARCIA ET AL.
342
E
A
MOB
I
./
+
\I I
\I
E
C
D n
Fig. 3. Camera lucida drawing of seven transverse sections through the brain of a lizard that received a restricted HRP injection (B) in the AOB. Retrograde labeling is indicated by open circles; fiber and terminal-like labeling are represented by lines and dots, respectively.
Contralateral intermediate olfactory tract. The contralateral telencephalic hemisphere contained labeled somata in the ventral part of the AON. Their axons coursed rostralward through the iot and stria medullaris, and crossed the midsagittal plane via the commissura habenu-
laris. In the injected hemisphere, labeled fibers of the stria terminalis entered the aot directly at the level of the ventral amygdala (Figs. 3F, 4E). Ipsilateral intermediate olfactory tract. Labeled neurons in the ipsilateral diagonal band nucleus (Fig. 3D)
LIZARD OLFACTORY BULB CONNECTIONS
Fig. 4. A. Terminallike labeling observed in outer half of layer 1in the lateral cortex (L) of a lizard that received a restricted HRP injection in the contralateral MOB. Bar = 150 pm. B. High power microphotograph of the ventrolateral amygdaloid nucleus (VL)of a lizard that received a HRP injection into the ipsilateral MOB. Arrowheads point to varicosities of anterogradely labeled axons. Bar = 10 pm. C. Retrogradely labeled cells in the ipsilateral DBN after HRP injection into the ipsilateral MOB. Some of the labeled neurons seem to be partially immersed in the intermediate olfactory tract that also shows intense fiber labeling. Bar = 150 pm. D. Low power microphotograph of the accessory olfactory bulb after HRP injection in the ipsilateral main olfactory bulb. Labeled somata (arrowheads) and terminals are ob-
343
served in the mitral layer. Terminallike labelling is also observed in part of the granular stratum (gl). Bar = 100 pm. E. Intense terminallike labeling observed in the amygdaloid formation of a specimen that received an HRP injection into the ipsilateral AOB. Retrogradely labeled cells can be observed in the mural layer of the nucleus sphericus, whereas those in the hilus are masked by the intense anterograde labeling that occupies this zone. Some labeled fibers (arrows) enter the amygdaloid terminal field from the stria medullaris. Bar = 150 pm. F. Retrogradely labeled mitral cells (arrows) in the MOB after HRP injection into the ipsilateral AOB. Terminallike labeling is observed in the mitral cell layer, inner plexiform layer and the outer rim of the granular stratum. Bar = 10 pm.
F. MARTINEZ-GARCIA ET AL.
344 sent their axons rostralward through the iot, which joined the aot at retrobulbar levels. Retrograde labeling in the DBN extended into the lateral preoptic area (Fig. 3E). Main olfactory bulb. The MOB displayed labeling of cell bodies in the mitral cell layer and terminallike labeling in the mitral cell layer, inner plexiform layer, and the outer half of the granular layer (Figs. 3A,4F). Mesencephalon. Relatively large but restricted HRP injections into the AOB resulted in bilateral labeling of a few somata in the locus coeruleus. Retrograde labeling was also found in the midbrain raphe nucleus (Fig. 3G).
HRP injections into the rostral lateral cortex The injections of HRP into the rostral lateral cortex affected as well the lateral olfactory tract. In these cases the MOB showed bilateral labeling of mitral cells (with ipsilatera1 predominance) and ipsilateral terminallike labeling in the inner plexiform layer and mitral layer (Fig. 5A).
HRP deposits on the amygdala Deposits of HRP-saponin crystals in the dorsal amygdala (including the dorsolateral and central nuclei and the dorsal half of the nucleus sphericus) resulted in retrograde labeling of mitral cells in the AOB. Moreover, terminallike labeling was observed in the granular layer (Fig. 5B).
DISCUSSION Efferent connections of the olfactory bulbs Projections o f the nzain Olfactory bulbs. The MOB projects to a series of superficial structures of both telencephalic hemispheres, such as the lateral cortex, the medial part of the AON, the rostral septum, the olfactory tubercle, the nucleus of the lateral olfactory tract, and the ventrolateral amygdala. These superficial structures show a trilaminar organization that is accompanied by a laminar distribution of their afferents. The outer half of their outer molecular layer (la) is occupied by the ipsilateral and contralateral olfactory projections, whereas the inner half of layer 1 (lb), the cell layer (2), and the inner molecular layer (3) receive, at least in the case of the lateral cortex and the extern amygdala, commissural and intrinsic connections (MartinezGarcia et al., '86; Martinez-Garcia, '88; Martinez-Garcia and Lorente, '88). Studies on the olfactory connections of crocodiles, squamate reptiles, turtles, and birds (Scalia et al., '69; Halpern, '76, '80; Ulinski and Peterson, '81; Reiner and Karten, '85; Lohman et al., '88; this study) have described a similar pattern of olfactory projections to the telencephalic hemispheres. Hence it appears that the olfactory system has been highly conservative during reptilian evolution. Other researchers have reported that the MOB projection is restricted to the rostral pole of the lateral cortex, based on results of experiments using silver impregnation of degenerating axoplasm (Gamble, '52 in Lacerta; Ulinski and Peterson, '81 in Dipsosaurus). Morever, Halpern (1976) using similar techniques, did not find in snakes a MOB projection to the medial retrobulbar formation through the medial olfactory tract. The close taxonomic proximity between Lacerta and Podarcis (the genus Podarcis was part of the old genus Lacerta) suggests that differences between our data and those of Gamble ('52) are probably due to limitations of the early silver impregnation methods. However, modern lesiondegeneration tracing techniques, such as those used by Ulinski and Peterson ('81) and Halpern ('76) yield results
Fig. 5. A. Retrogradely labeled mitral cells (arrows) in the MOB after HRP injection into the ipsilateral rostral lateral cortex. Diffuse terminallike labeling appears in the mitral cell and inner plexiform layers. B. Retrograde (thin arrows) and anterograde (thick arrows) labeling observed in the AOB after HRP injection into the ipsilateral amygdala, involving the dorsolateral amygdaloid nucleus and the nucleus sphericus. Bars = 100 p m (A),200 p m (B).
similar to those obtained through intraaxonic transport of tracers (compare our results with those of Lohman et al., '88 in Gekko). Thus it seems that the discrepancy between results of previous workers and our own may be due to species differences in the organization of the olfactory system among squamate reptiles, rather than to the use of different tract-tracing techniques. Further studies in other lizards and snakes are needed to understand the physiological and/or comparative meaning of such differences. The presence of an accessory olfactory system does not imply a decrease of the MOB projection to the amygdala. In fact, Podarcis shows a well-developed accessory olfactory system (see Fig. 1) together with an extensive bilateral projection of the MOB to the amygdaloid formation (similar to that found in crocodiles, birds, and turtles). These facts strongly support the dual olfactory hypothesis (Raisnian, '72; Scalia and Winans, '75); if both the vomeronasal and
LIZARD OLFACTORY BULB CONNECTIONS olfactory systems were involved in different behavioral domains, the reduction or absence of one of these systems would be a consequence of the 100sof its domain but should have no effect on the development and function of the other one. Our HRP injections into the lateral cortex indicate that the cells-of-origin of the MOB centripetal projections are the mitral cells, as suggested earlier by Golgi studies in the same species (Garcia-Verdugo et al., '87). Projections o f the accessory olfactory bulb. Besides the possibility of a direct projection to several structures embedded in the accessory olfactory tract, such as the ventral part of the AON and the nucleus of the accessory olfactory tract (belonging to the perifascicular complex of Ulinski and Peterson, %I), the AOB projects to the ipsilateral nucleus sphericus, the central and ventromedial amygdaloid nuclei, and to the lateral part of the bed nucleus of the stria terminalis. Our results confirm recent data in Gekko (Lohman et al., '88) according to which the projection from the AOB to the amygdala is more extensive than previously believed, at least in some lacertilian species. The relatively reduced AOB projection to the amygdala in other lizards (Ulinski and Peterson, '81) and snakes (Halpern, '76) may reflect some heterogeneity among squamate reptiles. Further data are required to clarify this issue.
Interaction between the vomeronasal and olfactory systems. The segregation of both olfactory systems in squamate reptiles is well documented (Halpern, '76; Ulinski and Peterson, '81; Lohman et al., '88). However, our results indicate that both systems apparently converge onto the AON and the amygdala. Although no obvious overlapping was found between the MOB and AOB projections within these structures, only studies of their intrinsic connections and electrophysiology can clarify whether the two chemosensory modalities are processed separately or not. In the hamster, a mammal, Licht and Meredith ('87) found convergence of main and accessory inputs onto single neurons of the vomeronasal amygdala (posteromedial cortical nucleus). An unexpected finding of this work has been the observation of a reciprocal connection between the main and accessory olfactory bulbs. These connections arise from the mitral cells (see Fig. 4D,F) and terminate in the mitral cell layer, inner plexiform layer and part of the granular layer in both olfactory bulbs, providing a direct feedback loop between the MOB and AOB. The nature and meaning of this interrelation is not known, but it strongly suggests that an intimate coordination of the vomeronasal and olfactory systems at this level may be necessary for the performance of complex chemoreception-related tasks. So far, a similar projection (from the MOB to the AOB) has been described only for an amphibian (Kemali and Gugliemotti, '87).
Centrifugal connections of the olfactory bulbs According to our results, the olfactory bulbs are reciprocally connected with the main targets of their centripetal connections. The vomeronasal amygdala, including the nucleus of the accessory olfactory tract, the central and ventromedial amygdaloid nuclei, the nucleus sphericus, and the bed nucleus of the stria terminalis project to the ipsilateral AOB. The contralateral nucleus sphericus (hilus) projects to the AOB through the anterior commissure, and the vomeronasal AON (ventromedial part) projects bilaterally to the AOB. The contralateral fibers course through the stria medullaris-habenular commissure system.
345
The MOB receives bilateral input from the rostral lateral cortex (olfactory cortex) and olfactory AON (dorsomedial part) (the contralateral component coursing through the striae medullaris-habenular commissure system). The reciprocal connections between the olfactory bulbs and their telencephalic target structures may provide feedback loops allowing amplification of the chemosensory inputs. In mammals (Gray et al., '87; Gray and Skinner, '88) epileptiform activity in the MOB elicited by odorant stimuli is mediated and maintained by centrifugal afferents from the olfactory cortex, suggesting that a feedback circuit between both structures is involved. In P. hispanica, these centrifugal projections terminate in the deep (AOB) or superficial (MOB) granular layer. According to Golgi studies (Garcia-Verdugo et al., '87; Llahi and Garcia Verdugo, '891, these zones are occupied by dendritic branches of granular and mitral cells. In addition to these "specific" afferents, both olfactory bulbs share some other sources of afferents in the forebrain: the diagonal band nucleus, the lateral preoptic area, and the caudal dorsal cortex. The projection from the dorsal cortex has been found in Gekko to arise from the rostral third of this cortical area (Hoogland and Vermeulen Van der Zee, '89), whereas our results clearly indicate that it originates in its caudal half. This discrepancy may be attributed to interspecific variance in the organization of the cerebral cortex. The dorsal cortex and the DBN are the targets for nonolfactory aiTerents: the dorsal cortex receives multimodal afferents from the dorsolateral anterior thalamus (Belekhova and Ivazov, '83; Lohman and Van WoerdenVerkley, '78; Bruce and Butler, '84; Martinez-Garcia and Olucha, '88; Martinez-Garcia and Lorente, '90); the DBN is reached by ascending projections from the hypothalamus, raphe nucleus, and other mesencephalic structures (Martinez-Garcia, '88). Hence, it is not likely that these structures participate in processing the chemosensory information; instead they should modulate or modify the olfactory and vomeronasal input depending on the general physiological status of the organism. A similar role for the DBN centrifugal projection has been suggested by Price and Powell ('70) in the rat. Finally, some extratelencephalic nuclei project to both olfactory bulbs, namely, the midbrain raphe nucleus and the locus coeruleus. Gathering together our results and the available data on the serotonergic (Smeets and Steinbusch, '88) and noradrenergic (Smeets and Steinbusch, '89) systems of the Gekko forebrain and midbrain, it can be concluded that the raphe nucleus and locus coeruleus are responsible, respectively, for the serotonergic and noradrenergic innervation of the olfactory bulbs. Our restricted injections in the olfactory bulbs labeled only a few (2-3) neurons in those nuclei. In fact, labeling of locus coeruleus cells was observed only after a relatively large injection into the AOB. However, noradrenergic innervation of the MOB has also been described, suggesting that it actually receives a projection from the locus coeruleus, which may be too diffuse and scarce for being detected by injections of small HRP volumes.
Comparative remarks Our results show that the hodological organization of the olfactory and vomeronasal systems is similar in mammals and lizards, although some differences are observed. The most striking of these is the complete absence in mammals of contralateral olfactory projections (for review, see Swit-
346
zer et al., '85). This feature is parallelled by the absence of commissural connections from telencephalic structures through the stria medullaris-habenular commissure system, which are present in the rest of tetrapoda (Reiner and Karten, '85; Kemali and Gugliemotti, '87). Instead, mammalian olfactory structures projecting contralaterally (AON and pyriform cortex) use the anterior commissure as a route. The MOB in both mammals and reptiles connects reciprocally with the AON (mammals: Broadwell, '75; Scalia and Winans, '75; Dennis and Kerr, '76; Skeen and Hall, '77; De Olmos et al., '78; Shipley and Adamek, '84; De Carlos et al., '89). In mammals, another retrobulbar structure reciprocally connected with the MOB is the subfield tt, of the taenia tecta or precommissural hippocampus, the reptilian counterpart of which may be the dorsomedial part of the AON as defined by us. In fact, the AONm is a rostral extension of the medial cortex (a similar structure in Dipsosaurus was called 'rostral rudiment of the medial cortex' by Ulinski and Peterson, '81) that has been considered as a reptilian homologue of the mammalian hippocampus (Curwen, '37; Goldby and Gamble, '57; Northcutt, '67; Ebbesson and Voneida, '69; Lohman and Mentink, '72). The mammalian olfactory cortex is composed of prepiriform, piriform, and entorhinal areas (Heimer, '69; Scalia and Winans, '75; Skeen and Hall, '77). Its rostral part (prepiriform cortex) projects back bilaterally to the olfactory bulb (Dennis and Kerr, '76; De Olmos et al., '78; De Carlos et al., '89). Likewise, the rostral part of the lacertilian olfactory (lateral) cortex projects bilaterally to the MOB. However, this rostral part of the lateral cortex of lizards projects as well to the 'hippocampal formation' (medial cortex) (Lohman and Mentink, '72; MartinezGarcia et al., '86) like the entorhinal cortex of mammals does (Schwerdtfeger, '84).This fact suggests that, like the visual system (Ebbesson, '811, the olfactory system has undergone a process of parcelation during vertebrate evolution. In mammals (Broadwell and Jacobowitz, '76; Dennis and Kerr, '76; De Olmos et al., '78) and reptiles (results of this work), the main centrifugal afferent of the MOB arises from the DBN. Moreover, some cells of the lacertilian lateral preoptic area caudally adjacent to the DBN project to the MOB; this cell group may be, according to their projections and location, the reptilian counterpart of the mammalian substantia innominata (De Olmos et al., '78). The presence of acetylcholinesterase reactive cells in this position has been recently reported in the tegu lizard (Follett, '89). A projection from the DBN and 'substantia innominata' to the AOB has been found in reptiles (this work) and in some mammals by Broadwell and Jacobowitz ('76). Although these authors interpreted their results as due to spread of injected HRP into the MOB, our results on that point are conclusive, since the MOB and AOB of Podarcis are large enough for restricting injections into either one or the other. The projection of the MOB to superficial structures immersed in the olfactory tract (olfactory tubercle and nucleus of the lateral olfactory tract) is also found in mammals (Broadwell, '75; Scalia and Winans, '75; Skeen and Hall, '77). Concerning the connections of the AOB, the high similarity of the efferent connections in mammals and reptiles has been long discussed by other authors (Halpern, '76; Ulinski and Peterson, '81). Our results support findings of Lohman
F. MARTINEZ-GARCIA ET AL. et al. ('88) according to which there is in reptiles, as in mammals (Broadwell, '75; Skeen and Hall, '771, a direct projection from the AOB to part of the bed nucleus of the stria terminalis. Moreover, the nucleus sphericus, previously compared to the posteromedial cortical nucleus of the mammalian amygdala (Halpern, '76; Ulinski and Peterson, '81) in view of its massive afferent from the AOB, is the origin of a bilateral (with ipsilateral predominance) centrifugal projection to the AOB, like the mammalian posteromedial cortical nucleus of the amygdala (Broadwell and Jacobowitz, '76; De Olmos et al., '78). Likewise, the bed nucleus of the stria terminalis and the bed nucleus of the accessory olfactory tract project to the AOB in reptiles (this work) as well as in mammals (De Olmos et al., '78). The centrifugal connection of the AON to the AOB described in this work has not been observed in mammals (Switzer et al., '85). Our results show that this labeled cell group is a rostral extension of the bed nucleus of the accessory olfactory tract (projecting in mammals to the AOB, De Olmos et al., '78).Hence, this part of the AON may be considered as the rostral tip of the amygdaloid formation, which would reflect the high development that the vomeronasal system has reached in squamate reptiles (Halpern, '87). On the whole, the fiber connections traced in this study hint at many putative homologies of the olfactory system in the brain of reptiles and mammals. However, conclusive data will be obtained only after the investigation of a larger sample of species than those studied so far.
ACKNOWLEDGMENTS This work has been supported in part by a grant from "Accions concertades d'investigacio de 1'Universitat de Valencia" project number 5183.
LITERATURE CITED Belekhova, M.G., and N.I. Ivazov (1983) Analysis of transmission of visual, somatic, and audiovibrational sensory information to the hippocampal cortex of lizards. Neirofiziol. 15:153-159. Bellairs, A. (1969) The Life of Reptiles. London: Weidenfield and Nicholson, 590 pp. Broadwell, R.D. (1975) Olfactory relationships of the telencephalon and diencephalon of the rabbit. I. An autoradiographic study of the efferent connections of the main and accessory olfactory bulbs. J. Comp. Neurol. 163:329-346. Broadwell, R.D., and D.M. Jacobowitz (1976) Olfactory relationships of the telencephalon and diencephalon in the rabbit. 111. The ipsilateral centrifugal fibers to the olfactory bulbar and retrobulbar formations. J. Comp. Neurol. 170:321-346. Bruce, L., and A.B. Butler (1984) Telencephalic connections in lizards. I. Projections to cortex. J. Cornp. Neurol. 229.585-601. Burghart, G. (1970) Chemical perception in reptiles. In J.W. Johnston, D.G. Moulton and A. Turk (eds): Communication by Chemical Signals. New York: Appleton-Century-Crofts, pp. 24 1-308. Curwen, A.O. (1937) The telencephalon of Tupinambis nigropunctatus. I. Medial and cortical areas. J. Comp. Neurol. 02375-404. Curwen, A.O. (1939) The telencephalon of Tupinanbis nigropunctatus. 111. Arnygdala. J. Comp. Neurol. 713313-636. De Carlos, J.A., L. Lopez-Mascaraque, and F. Valverde (1989) Connections of the olfactory bulb and nucleus olfactorius anterior in the hedgehog (Erinaceus europaeus): Fluorescent tracers and HRP study. J. Comp. Neurol. 279:601-618. De Olmos, J.S., H. Hardy, and L. Heirner (1978) The afferent connections of the main and accessory bulb formations in the rat: An experimental HRP study. J. Cornp. Neurol. 181r213-244. Dennis, B.J., and D.B. Kerr (1976) Origin of the olfactory bulb centrifugal fibers in the cat. Brain Res. 110:593-600.
LIZARDOLFACTORYBULB CONNECTIONS Ebbesson, S.O.E. (1981) Evolution and ontogeny of neural circuits. Behav. Brain Sci. 7,321-366. Ebbesson, S.O.E., and T.J. Voneida (1969) The cytoarchitecture of the tegu lizard Tupinambis nigropunctatus. Brain Behav. Evol. 2:431466. Follett, K.A. (1989) A telencephalospinal projection in the tegu lizard (Tupinambrs teguixin). Brain Res. 496:89-97. Gabe, M., and H. St. Girons (1976) Contribution a la morphologie comparee des fosses nasales et de leurs annexes chez les lepidosauriens. Mem. Mus. Nat. D‘Hist. Nat. Ser. A. 98:l-87. Gamble, H.J. (1952) An experimental study on the secondary olfactory connections in Lacerta uiridis. J. Anat. 86:180-196. Garcia-Verdugo, J.M., S. Llahi, I. Farinas, and V. Martin (1987) Laminar organization of the main olfactory bulb ofPodarcis hispanica: an electron microscopic and Golgi study. J. Hirnforsch. 27:87-100. Goldby, R., and H.J. Gamble (1957)The reptilian cerebral hemispheres. Biol. Rev. 32,383420. Gray, C.M., and J.E. Skinner (1988) Centrifugal regulation of neuronal activity in the olfactory bulb of the waking rabbit as revealed by reversible cryogenic blockade. Exp. Brain Res. 69:378-386. Gray, C.M., W.J. Freeman, and J.E. Skinner (1987) Induction and maintenance of epileptiform activity in the rabbit olfactory bulb depends on centrifugal input. Exp. Brain Res. 68:210-212. Halpern, M. (1976) The efferent connections of the olfactory bulb and accessory olfactory bulb in the snakes Thamnophis sirtalis and Thamnophis radix. J. Morphol. 150:553-578. Halpern, M. (1980) The telencephalon of snakes. In S.O.E. Ebbesson (ed): Comparative Neurology of the Telencephalon. New York: Plenum Press, pp. 257-294. Halpern, M. (1987) The organization and function of the vomeronasal system. Ann. Rev. Neurosci. lOt325-362. Heimer, L. (1969)The secondary olfactory connections in mammals, reptiles and sharks. Ann. N.Y. Acad. Sci. 16729-146. Hoogland, P.V., and E. Vermeulen-Van der Zee (1989) Efferent connections of the dorsal cortex of the lizard Gekko gecko studied with Phaseolus vulgaris-leucoagglutinin. J. Comp. Neurol. 285289-303. Kemali, M., andV. Gugliemotti (1987) A horseradish peroxidase study of the olfactory system of the frog, Rana esculenta. J. Comp. Neurol. 263:400417. Licht, G., and M. Meredith (1987) Convergence of main and accessory olfactory pathways onto single neurons in the hamster amygdala. Exp. Brain Res. 69:7-18. Llahi, S., and J.M. Garcia-Verdugo (1989) Neuronal organization of the accessory olfactory bulb of the lizard Podarcis hispanica: Golgi study. J. Morphol. 202:13-28. Lohman, A.H.M., and I. Van Woerden-Verkley (1978) Ascending connections to the forebrain in the tegu lizard. J. Comp. Neurol. 182:555-594. Lohman, A.H.M., and G.M. Mentink (1972) Some cortical connections of the tegu lizard (Tupinambisteguixin). Brain Res. 45:325-344. Lohman, A.H.M., P.V. Hoogland, and R.J.G.M. Witjes (1988) Projections from the main and accessory olfactory bulbs to the amygdaloid complex in the lizard Gekko gecko. In W.K. Schwerdtfeger and W.J.A.J. Smeets (eds): The Forebrain of Reptiles. Current Concepts of Structure and Function. Base1 Karger, pp. 41-49. Martinez-Garcia, F. (1988) Conexiones de las ireas corticales y subcorticales del telencefalo de la lagartija comdn Podarcis hispanica. Doctoral dissertation, University of Valencia.
347 Martinez-Garcia, F., and M.J. Lorente (1988) Fibre connections of the amygdala in the lizard, Podarcis hispanica. 11th Meeting of the E.N.A., Satellite Symposium, “Comparative aspects on the structure and development of the forebrain in lower vertebrates.” Zurich, Switzerland. Martinez-Garcia, F., and M.J. Lorente (1990) Thalamo-cortical projections in the lizard Podarcis hispanica. In W.K. Schwerdtfeger and P. Germroth (eds): The Forebrain in nonmammals. New Aspects of Structure and Development. Berlin: Springer-Verlagpp. 93-102. Martinez-Garcia, F., and F.E. Olucha (1988) Afferent projections to the Timm-positive cortical areas of the telencephalon of lizards. In W.K. Schwerdtfeger and W.J.A.J. Smeets (eds): The Forebrain of Reptiles. Current Concepts of Structure and Function. Base1 Karger, pp. 30-40. Martinez-Garcia, F., M. Amiguet, F. Olucha, and C. L6pez-Garcia (1986) Connections of the lateral cortex in the lizard Podarcis hispanica. Neurosci. Lett. 63:39-44. Northcutt, R.G. (1967)Architectonic studies of the telencephalon of Iguana iguana.J. Comp. Neurol. 130:109-148. Olucha, F., F. Martinez-Garcia, and C. L6pez-Garcia (1985) A new stabilizing agent for the tetramethyl benzidine (TMB) reaction product in the histochemical detection of horseradish peroxidase (HRP). 3. Neurosci. Meth. 13t131-138. Price, J.L., and P.J. Powell (1970) An experimental study of the origin and the course of the centrifugal fibers to the olfactory bulb in the rat. J. Anat. 107t215-237. Raisman, G. 11972)An experimental study of the projection of the amygdala to the accessory olfactory bulb and its relationship to the concept of a dual olfactory system. Exp. Brain Res. 14:395408. Reiner, A,, and H.G. Karten (1985) Comparison of the olfactory bulb projections in pigeon and turtles. Brain Behav. Evol. 27t11-27. Scalia, F., M. Halpern, and W. Riss (1969) Olfactory bulb projections in the South American caiman. Brain Bebav. Evol. 2:238-262. Scalia, F., and S.S. Winans (1975) The differential projections of the olfactory bulb and accessory olfactory bulb in mammals. Brain Res. 161:31-56. Scbwerdtfeger, W.K. (1984) Structure and Fiber Connections of the Hippocampus: A Comparative Study. Berlin: Springer-Verlag, 74 pp. Shipley, M.T., and G.D. Adamek (1984) The connections of the mouse olfactory bulb: a study using orthograde and retrograde transport of wheatgerm agglutinin conjugated to horseradish peroxidase. Brain Res. Bull. 12:669-688. Skeen, L.C., and W.C. Hall (1977) Efferent projections of the main and the accessory olfactory bulb in tree shrew (Tupaia glis). J. Comp. Neurol. 172.1-36. Smeets, W.J.A.J., and H.W.M. Steinbusch (1988) Distribution of serotonin immunoreactivity in the forebrain and midbrain of the lizard Gekko gecko. J. Comp. Neurol. 271:419-434. Smeets, W.J.A.J., and H.W.M. Steinbusch (1989)Distribution of noradrenaline immunoreactivity in the forebrain and midbrain of the lizard Gekko gecko. J. Comp. Neurol. 285t453-466. Switzer, R.C., J. De Olmos, and L. Heimer (1985) Olfactory system. In G. Paxinos (ed): The Rat Nervous System. Sydney: Academic Press, pp. 1-36. Ulinski, P.S. (1983) Dorsal ventricular ridge: A Treatise on Forebrain Organization in Reptiles and Birds. New York: John Wiley & Sons. Ulinski, P.S., and E.H. Peterson (1981) Patterns of olfactory projections in the desert iguana, Dipsosaurus dorsalis. J. Morphol. 168:189-227.
Afferent and Efferent Connections of the Olfactory Bulbs in the Lizard Podarcis hispanica FERNANDO MARTINEZ-GARCIA, FRANCISCO E. OLUCHA, VICENT TERUEL, MARIA J. LORENTE, AND WALTER K. SCHWERDTFEGER Universitat de Valencia, Facultat de Cikncies Biolbgiques, Departament de Biologia Animal. Unitat de Morfologia Microscbpica. Spain (F.M.-G., F.E.O., V.T., M.J.L.),and Max-Planck Institut fur Hirnforschung, Frankfurt am Main, Germany (W.K.S.)
ABSTRACT The connections of the olfactory bulbs of Podarcis hispanica were studied by tract-tracing of injected horseradish peroxidase. Restricted injections into the main olfactory bulb (MOB) resulted in bilateral terminallike labeling in the medial part of the anterior olfactory nucleus (AON) and in the rostral septum, lateral cortex, nucleus of the lateral olfactory tract, and ventrolateral amygdaloid nucleus. Bilateral retrograde labeling was found in the rostral lateral cortex and in the medial and dorsolateral AON. Ipsilaterally the dorsal cortex, nucleus of the diagonal band, lateral preoptic area, and dorsolateral amygdala showed labeled cell bodies. Retrogradely labeled cells were also found in the midbrain raphe nucleus. Results from injections into the rostral lateral cortex and lateral olfactory tract indicate that the mitral cells are the origin of the centripetal projections of the MOB. Injections in the accessory olfactory bulb (AOB) produced ipsilateral terminallike labeling of the ventral AON, bed nucleus of the accessory olfactory tract, central and ventromedial amygdaloid nuclei, medial part of the bed nucleus of the stria terminalis, and nucleus sphericus. Retrograde labeling of neurons was observed ipsilaterally in the bed nucleus of the accessory olfactory tract and stria terminalis, in the central amygdaloid nucleus, dorsal cortex, and nucleus of the diagonal band. Bilateral labeling of somata was found in the ventral AON, the nucleus sphericus (hilus), and in the mesencephalic raphe nucleus and locus coeruleus. Injections into the dorsal amygdala showed that the mitral neurons are the cells of origin of the AOB centripetal projections. Reciprocal connections are present between AOB and MOB. To our knowledge, this is the first study to address the afferent connections of the olfactory bulbs in a reptile. On the basis of the available data, a discussion is provided of the similarities and differences between the reptilian and mammalian olfactory systems, as well as of the possible functional role of the main olfactory connections in reptiles. Key words: comparative neuroanatomy,HRP, reptile, amygdala, vomeronasal system, olfactory system
In amphibians, squamate reptiles (lizards and snakes), and the majority of mammals, chemical signals enter the telencephalon in two different ways: from the nasal epithelium via the main olfactory bulbs (MOB), and from the vomeronasal organ via the accessory olfactory bulbs (AOB). Experimental studies on the behavior of reptiles (for review, see Burghart, ’70, Halpern, ’87) indicate that this kind of information (chemosensory signals from both the olfactory and vomeronasal systems) is essential for these animals in several biologically significant activities, such as sexual behavior and foraging. Moreover, the olfactory structures of the brain, especially those of the vomeronasal system, are especially well developed in snakes and terrestrial lizards (Bellairs, ’69; Gabe and Saint Girons, ’76).
o 1991 WILEY-LISS, INC.
Other anatomical features accompany the high development of the accessory olfactory system in those reptiles, namely, the presence of a forked tongue useful to introduce “odorant” particles into the Jacobson’s organ. In spite of the importance of olfaction for the biology of reptiles, anatomical investigations on fiber connections of their olfactory bulbs have so far been restricted to the study of their centripetal projections, using anterograde degeneration techniques (Gamble, ’52; Heimer, ’69; Halpern, ’76; Accepted October 31,1990 Address reprint requests to Fernando Martinez-Garcia, Dept. Biologia Animal, Unitat de Morfologia Microscopica. Fac. C. Biologiques, Univ. Valencia. C. Dr. Moliner, 50. E-46100 Burjassot, Spain.
F. MARTINEZ-GARCIA E T AL.
338
Ulinski and Peterson, ’81; Lohman et al., ’88),anterograde transport of tritiated aminoacids (Reiner and Karten, ’851, and Phaseolus uulgaris leucoagglutinin (Lohman et al., ’88). The knowledge of the olfactory bulb afferents should be equally essential for understanding the structure and the physiology of the vomeronasal and olfactory systems. Hence, in the present study, anterograde and retrograde transport of horseradish peroxidase (HRP) were used in order to: (1)map the origin of afferent projections to the bulbs, and (2) check the efferents of the olfactory bulbs with a tracing technique different from those that have been previously applied.
MATERIAL AND METHODS For this study 35 adult specimens of the lizard Podarcis hispanica (45-60 mm snout-vent length) were used. Under ether anaesthesia, 10-20 nl of 20% HRP (Sigma, type VI) in 2% dimethyl sulfoxide or 10% Saponin (Merck, Darmstadt, F.R.G.) were injected in the main olfactory bulb (MOB) of 16 specimens, and in the accessory olfactory bulb (AOB) of 11lizards. Additional HRP injections were performed in the rostral lateral cortex (4 specimens) and in the amygdala (dorsolateral amygdaloid nucleus and nucleus sphericus; 4 lizards). After 7-9 days survival a t room temperature (18-2loC), animals were anaesthetized again and were transcardially perfused with saline followed by cold (4°C) fixative containing 1.25% glutaraldehyde and 1% paraformaldehyde in 0.1 M phosphate buffer, pH 7.2. Brains were immersed in the same fixative for 4 hours and rinsed overnight at 4°C in 30% phosphate-buffered sucrose. Fifty-pm-thick frontal sections of the brains were obtained using a freezing microtome. Sections were processed according to the tetramethyl benzidine-ammonium heptamolybdate method for histochemical detection of peroxidase activity (Olucha et al., ’85), mounted in chromalumcoated slides, and counterstained with neutral red.
RESULTS Prior to reporting the results of HRP injections, we describe briefly the organization of relevant regions of the telencephalic hemispheres in Podarcis. The terminology
employed for this and for the hodological study allows us to compare our results for Podarcis with those of other authors for different reptilian species.
Terminology The rostral tip of the telencephalic hemisphere plus the olfactory peduncle are known as the “retrobulbar formation” (RBF). Within the RBF two superficial structures showing a laminar arrangement can be distinguished: on the one hand, the lateral cortex, that is, the rostralmost cortical area and, on the other hand, the ventral olfactory tubercle (OT) (Fig. lA,B). The “core” of the RBF is occupied by a complex structure that we have called, according to the classical nomenclature, anterior olfactory nucleus (AON). Three different subnuclei are distinguished within the AON with respect to neuronal packing and topographical position (Fig. 1A,B). The laterodorsal AON (AON1) is formed by a group of scattered neurons just below the lateral cortex. It displays a large rostrocaudal extent, being evident between the caudal margin of the olfactory bulbs and the rostral margin of the dorsal cortex and pallial thickening. These features resemble those of the region called “polymorphic layer of the dorsal cortex” by Halpern (’80). The dorsomedial AON (AONm) (Fig. 1B) displays a high density of neuronal packing. It coincides in location and neuronal density with the dorsomedial retrobulbar formation of Halpern (’80). Moreover, it is caudally bounded by the medial cortex showing a diffuse trilaminar organization; for this reason a similar region in the rostral telencephalon of Dipsosaurus dorsalis was called by Ulinski and Peterson (’81) “rostral rudiment of the medial cortex.” Finally, the ventral AON (AONv) (Fig. 1A) is made up of a compact group of juxtaependymal neurons, caudally bounded by the rostral ADVR. It roughly resembles the retrobulbar formation of Ulinski and Peterson (’81). The olfactory tubercle is rostrally adjacent to the nucleus of the diagonal band (DBN) (Fig. 1C). The limit between both structures is rather diffuse in a Nissl preparation, but it is clear in terms of fiber connectivity, e.g., only the DBN projects to the cerebral cortex (Bruce and Butler, ’84; Martinez-Garcia et al., ’86). The DBN reaches the caudal tip of the telencephalon, up to diencephalic levels (caudally
Abbreviations
ac ACC
ADVR AOB aot AON AONl AONm AONv BAOT BNM BST CAN D DEN DLAN
DM gl HRP ipl iot
anterior commisure nucleus accumbens anterior dorsal ventricular ridge accessory olfactory bulb accessory olfactory tract anterior olfactory nucleus laterodorsal anterior olfactory nucleus dorsomedial anterior olfactory nucleus ventral anterior olfactory nucleus bed nucleus of the accessory olfactory tract bed nucleus of the medial forebrain bundle bed nucleus of the stria terminalis central amygdaloid nucleus dorsal cortex diagonal band nucleus dorsolateral amygdaloid nucleus dorsomedial cortex granular layer horseradish peroxidase inner plexiforrn layer intermediate olfactory tract
L LCO Irn lot LPA
M mcl MOB Mot mot Nlot NS OT
R RBF S srn STR
VL VM
lateral cortex locus coeruleus lateral forebrain bundle lateral olfactory tract lateral preoptic area medial cortex mitral cell layer main olfactory bulb main olfactory tract medial olfactory tract nucleus of the lateral olfactory tract nucleus sphericus olfactory tubercle midbrain raphe nucleus retrobulbar formation septum stria rnedullaris striatum ventrolateral amygdaloid nucleus ventromedial amygdaloid nucleus
LIZARD OLFACTORY BULB CONNECTIONS
Fig. 1. Nissl-stained transverse sections of the right telencephalic hemisphere of Podarcis hispanica showing the cytoarchitecture of the retrobulbar formation (A and B) amygdaloid formation (D and E)and other olfactory structures. Bar = 250 pm.
339
340
F. MARTINEZ-GARCIA ET AL.
Contralateral intermediate olfactory tract (iot). Fiber labeling appeared throughout the contralateral iot. At rostral levels this tract ascended in a midsagittal position through the rostral septum, where labeled fibers may perform synaptic contacts. In connection with this tract, labeled puncta and somata were observed in the AONm (Fig. 2C). The terminal-like labeling was restricted to the outer half of layer 1. Zpsilateral intermediate olfactory tract. Apparent labeling was present within the ipsilateral iot throughout its rostrocaudal extent. At rostral levels it occupied the outer layer of the olfactory tubercle where not only fiber labeling occurred but also intense terminallike labeling, suggesting the presence of synaptic contacts. Some smooth labeled fibers of the iot left the tract and entered the medial forebrain bundle at the level of either the nucleus accumbens or the nucleus of the medial forebrain bundle (Fig. 2D). The ipsilateral diagonal band nucleus showed retrograde labeling (see Fig. 4C) of neurons located mainly in the “horizontal limb.” They were usually fusiform with their main axis parallel to the pial surface. However, some stellate labeled neurons were also observed. This retrograde labeling continued beyond the caudal boundary of the DBN; several labeled neurons were found in the hypothalamic Injections of HRP into the main olfactory bulb lateral preoptic area (Fig. 2E), which sent their axons directly to the stria medullaris. After injections into the MOB, fiber labeling appeared Zpsilateral medial olfactory tract (mot). The mot takes bilaterally in the iot and lot, and in the ipsilateral mot (Fig. a midsagittal course giving rise to terminal fields in the 2). Contralateral connections were seen to course through rostral septum and in the AONm. Anterograde labeling the habenular commissure-striae medullaris system. In the showed the same laminar distribution as the contralateral following section we describe in detail the anterograde and side. Retrogradely labeled neurons were also observed in retrograde labeling found in connection with each labeled this part of the AON (Fig. 2 0 . tract. Accessory olfactory bulb. The ipsilateral accessory olfacContralateral lateral olfactory tract {lot). In the con- tory bulb showed terminallike labeling occupying the mitral tralateral hemisphere, the lateral cortex showed labeling of cell layer and part of the granular layer. Some retrogradely terminallike puncta and cell bodies (Fig. 2C). The terminal- labeled cells were observed in the mitral layer, indicating like labeling occupied the outer half of layer 1 (outer reciprocal connections between the MOB and AOB (Fig. 2B; plexiform layer) throughout its rostrocaudal axis (see Fig. see also Fig. 4D). 4A); a few retrogradely labeled somata were found in the Mesencephalon. At mesencephalic levels a few labeled lateral cortex at retrobulbar levels. Some neurons were also cells were found in the midbrain raphe nucleus (Fig. 2G). labeled in the AONl (Fig. 2C), whose axons were not detected. Since the nearest labeled tract to these neurons Injections of HRP into the accessory was the lot, this tract is likely to contain their axons. olfactory bulb Terminallike labeling was also observed in the so-called nucleus of the lot, in the lateral margin of the telencephalic After injections of HRP into the accessory olfactory bulb hemisphere. However, since this nucleus is immersed in the (AOB), intense fiber labeling was observed in the accessory lot, it is difficult to decide whether it actually corresponds to olfactory tract (aot). Some labeled axons were observed terminal labeling or to transversally cut fibers. Structures bilaterally in the intermediate olfactory tract (Fig. 3C,D,E) resembling boutons en passant were observed all along the and stria medullaris (Fig. 3F), as well as in the anterior tract and were especially frequent at the level of the (Fig. 3E) and habenular commissures. ventrolateral amygdala (see Fig. 4B). Zpsilateral accessory olfactory tract (aot). Reciprocal Zpsilateral lateral olfactory tract. The pattern of la- connections were observed between the AOB and the beled structures in connection with the ipsilateral lot was AONv, where retrograde and terminallike labeling was similar to that of the contralateral hemisphere, except for observed (Fig. 3 0 . Some labeled somata appeared in the the higher intensity of anterograde and fiber labeling and dorsal cortex, with the same distribution as seen after MOB the higher number of retrogradely labeled neurons. How- injections (Fig. 3D,E). ever, two main differences were noticed, namely retrograde The main target for the AOB centripetal connections is labeling of neurons in the dorsolateral amygdaloid nucleus the amygdaloid formation. At rostral levels, both retro(Fig. 2E), and in the cell layer of the caudal dorsal cortex grade and terminallike labeling was observed in the bed (Fig. 2D,E). At commissural levels, the medial and lateral nucleus of the accessory olfactory tract (Fig. 3C; belonging thirds of the dorsal cortex showed labeling, whereas the to the perifascicular complex of Ulinski and Peterson, ’81) intermediate portion remained unlabeled. The unlabeled and in the central amygdaloid nucleus. Caudally, a profuse intermediate portion decreased succesively at caudal levels. terminal field was found in the hilus of the nucleus Hence, at the caudal edge of the cortex, the whole dorsal sphericus, bordered by a narrow unlabeled band just below cortex showed labeled somata. the mural layer (Fig. 3E,F). This anterograde labeling
and ventrally to the anterior commissure), where it gradually looses its laminar organization and merges with the medial and lateral preoptic areas. The caudal edge of the ventrolateral telencephalon has been termed “archistriaturn” (e.g., Northcutt, ’67) and “basal dorsal ventricular ridge” (Ulinski, ’83). We prefer the classical terminology, or “amygdala” (Curwen, ’39). In Podarcis the most spectacular nucleus of the amygdala is the nucleus sphericus (Fig. 1E). A series of more rostrally located nuclei have been termed according to their topographical position: dorsolateral, ventrolateral, ventromedial, and central nuclei of the amygdala (Fig. 1D). At the levels of the latter nuclei, the ventral sulcus of the telencephalic ventricle is surrounded by a group of neurons crossed by the fibers of the stria terminalis. We therefore call it “bed nucleus of the stria terminalis” (Fig. 1D). Finally, our nomenclature of the olfactory tracts is based on Ulinski and Peterson (’81). We consider three different tracts related with the main accessory bulb (MOB); the lateral olfactory tract (lot),the medial olfactory tract (mot), and the intermediate olfactory tract (iot). The accessory olfactory tract (aot) connects the accessory olfactory bulb (AOB)with the telencephalic hemisphere.
LIZ ARDOLFACTORYBULBCONNECTIONS
341
iot Fig. 2. Schematic drawing of seven transverse sections of the brain of Podarcis showing the retrograde (open circles), fiber (lines)and terminallike labeling (dots) resulting from a restricted HRP injection (A) into the MOB.
extended into rostrally adjacent nuclei such as the ventromedial amygdaloid nucleus and the lateral part of the bed nucleus of the stria terminalis (Fig. 3F). Moreover, labeling of neuronal cell bodies was observed in these amygdaloid structures; in the nucleus sphericus labeled somata were
mainly located in the hilus but also in the mural layer (Fig. 4E). Finally, some retrogradely labeled hilar cells in the contralateral nucleus sphericus were seen to send their axons via the anterior commissure to the ipsilateral aot (Fig. 3E).
F. MARTINEZ-GARCIA ET AL.
342
E
A
MOB
I
./
+
\I I
\I
E
C
D n
Fig. 3. Camera lucida drawing of seven transverse sections through the brain of a lizard that received a restricted HRP injection (B) in the AOB. Retrograde labeling is indicated by open circles; fiber and terminal-like labeling are represented by lines and dots, respectively.
Contralateral intermediate olfactory tract. The contralateral telencephalic hemisphere contained labeled somata in the ventral part of the AON. Their axons coursed rostralward through the iot and stria medullaris, and crossed the midsagittal plane via the commissura habenu-
laris. In the injected hemisphere, labeled fibers of the stria terminalis entered the aot directly at the level of the ventral amygdala (Figs. 3F, 4E). Ipsilateral intermediate olfactory tract. Labeled neurons in the ipsilateral diagonal band nucleus (Fig. 3D)
LIZARD OLFACTORY BULB CONNECTIONS
Fig. 4. A. Terminallike labeling observed in outer half of layer 1in the lateral cortex (L) of a lizard that received a restricted HRP injection in the contralateral MOB. Bar = 150 pm. B. High power microphotograph of the ventrolateral amygdaloid nucleus (VL)of a lizard that received a HRP injection into the ipsilateral MOB. Arrowheads point to varicosities of anterogradely labeled axons. Bar = 10 pm. C. Retrogradely labeled cells in the ipsilateral DBN after HRP injection into the ipsilateral MOB. Some of the labeled neurons seem to be partially immersed in the intermediate olfactory tract that also shows intense fiber labeling. Bar = 150 pm. D. Low power microphotograph of the accessory olfactory bulb after HRP injection in the ipsilateral main olfactory bulb. Labeled somata (arrowheads) and terminals are ob-
343
served in the mitral layer. Terminallike labelling is also observed in part of the granular stratum (gl). Bar = 100 pm. E. Intense terminallike labeling observed in the amygdaloid formation of a specimen that received an HRP injection into the ipsilateral AOB. Retrogradely labeled cells can be observed in the mural layer of the nucleus sphericus, whereas those in the hilus are masked by the intense anterograde labeling that occupies this zone. Some labeled fibers (arrows) enter the amygdaloid terminal field from the stria medullaris. Bar = 150 pm. F. Retrogradely labeled mitral cells (arrows) in the MOB after HRP injection into the ipsilateral AOB. Terminallike labeling is observed in the mitral cell layer, inner plexiform layer and the outer rim of the granular stratum. Bar = 10 pm.
F. MARTINEZ-GARCIA ET AL.
344 sent their axons rostralward through the iot, which joined the aot at retrobulbar levels. Retrograde labeling in the DBN extended into the lateral preoptic area (Fig. 3E). Main olfactory bulb. The MOB displayed labeling of cell bodies in the mitral cell layer and terminallike labeling in the mitral cell layer, inner plexiform layer, and the outer half of the granular layer (Figs. 3A,4F). Mesencephalon. Relatively large but restricted HRP injections into the AOB resulted in bilateral labeling of a few somata in the locus coeruleus. Retrograde labeling was also found in the midbrain raphe nucleus (Fig. 3G).
HRP injections into the rostral lateral cortex The injections of HRP into the rostral lateral cortex affected as well the lateral olfactory tract. In these cases the MOB showed bilateral labeling of mitral cells (with ipsilatera1 predominance) and ipsilateral terminallike labeling in the inner plexiform layer and mitral layer (Fig. 5A).
HRP deposits on the amygdala Deposits of HRP-saponin crystals in the dorsal amygdala (including the dorsolateral and central nuclei and the dorsal half of the nucleus sphericus) resulted in retrograde labeling of mitral cells in the AOB. Moreover, terminallike labeling was observed in the granular layer (Fig. 5B).
DISCUSSION Efferent connections of the olfactory bulbs Projections o f the nzain Olfactory bulbs. The MOB projects to a series of superficial structures of both telencephalic hemispheres, such as the lateral cortex, the medial part of the AON, the rostral septum, the olfactory tubercle, the nucleus of the lateral olfactory tract, and the ventrolateral amygdala. These superficial structures show a trilaminar organization that is accompanied by a laminar distribution of their afferents. The outer half of their outer molecular layer (la) is occupied by the ipsilateral and contralateral olfactory projections, whereas the inner half of layer 1 (lb), the cell layer (2), and the inner molecular layer (3) receive, at least in the case of the lateral cortex and the extern amygdala, commissural and intrinsic connections (MartinezGarcia et al., '86; Martinez-Garcia, '88; Martinez-Garcia and Lorente, '88). Studies on the olfactory connections of crocodiles, squamate reptiles, turtles, and birds (Scalia et al., '69; Halpern, '76, '80; Ulinski and Peterson, '81; Reiner and Karten, '85; Lohman et al., '88; this study) have described a similar pattern of olfactory projections to the telencephalic hemispheres. Hence it appears that the olfactory system has been highly conservative during reptilian evolution. Other researchers have reported that the MOB projection is restricted to the rostral pole of the lateral cortex, based on results of experiments using silver impregnation of degenerating axoplasm (Gamble, '52 in Lacerta; Ulinski and Peterson, '81 in Dipsosaurus). Morever, Halpern (1976) using similar techniques, did not find in snakes a MOB projection to the medial retrobulbar formation through the medial olfactory tract. The close taxonomic proximity between Lacerta and Podarcis (the genus Podarcis was part of the old genus Lacerta) suggests that differences between our data and those of Gamble ('52) are probably due to limitations of the early silver impregnation methods. However, modern lesiondegeneration tracing techniques, such as those used by Ulinski and Peterson ('81) and Halpern ('76) yield results
Fig. 5. A. Retrogradely labeled mitral cells (arrows) in the MOB after HRP injection into the ipsilateral rostral lateral cortex. Diffuse terminallike labeling appears in the mitral cell and inner plexiform layers. B. Retrograde (thin arrows) and anterograde (thick arrows) labeling observed in the AOB after HRP injection into the ipsilateral amygdala, involving the dorsolateral amygdaloid nucleus and the nucleus sphericus. Bars = 100 p m (A),200 p m (B).
similar to those obtained through intraaxonic transport of tracers (compare our results with those of Lohman et al., '88 in Gekko). Thus it seems that the discrepancy between results of previous workers and our own may be due to species differences in the organization of the olfactory system among squamate reptiles, rather than to the use of different tract-tracing techniques. Further studies in other lizards and snakes are needed to understand the physiological and/or comparative meaning of such differences. The presence of an accessory olfactory system does not imply a decrease of the MOB projection to the amygdala. In fact, Podarcis shows a well-developed accessory olfactory system (see Fig. 1) together with an extensive bilateral projection of the MOB to the amygdaloid formation (similar to that found in crocodiles, birds, and turtles). These facts strongly support the dual olfactory hypothesis (Raisnian, '72; Scalia and Winans, '75); if both the vomeronasal and
LIZARD OLFACTORY BULB CONNECTIONS olfactory systems were involved in different behavioral domains, the reduction or absence of one of these systems would be a consequence of the 100sof its domain but should have no effect on the development and function of the other one. Our HRP injections into the lateral cortex indicate that the cells-of-origin of the MOB centripetal projections are the mitral cells, as suggested earlier by Golgi studies in the same species (Garcia-Verdugo et al., '87). Projections o f the accessory olfactory bulb. Besides the possibility of a direct projection to several structures embedded in the accessory olfactory tract, such as the ventral part of the AON and the nucleus of the accessory olfactory tract (belonging to the perifascicular complex of Ulinski and Peterson, %I), the AOB projects to the ipsilateral nucleus sphericus, the central and ventromedial amygdaloid nuclei, and to the lateral part of the bed nucleus of the stria terminalis. Our results confirm recent data in Gekko (Lohman et al., '88) according to which the projection from the AOB to the amygdala is more extensive than previously believed, at least in some lacertilian species. The relatively reduced AOB projection to the amygdala in other lizards (Ulinski and Peterson, '81) and snakes (Halpern, '76) may reflect some heterogeneity among squamate reptiles. Further data are required to clarify this issue.
Interaction between the vomeronasal and olfactory systems. The segregation of both olfactory systems in squamate reptiles is well documented (Halpern, '76; Ulinski and Peterson, '81; Lohman et al., '88). However, our results indicate that both systems apparently converge onto the AON and the amygdala. Although no obvious overlapping was found between the MOB and AOB projections within these structures, only studies of their intrinsic connections and electrophysiology can clarify whether the two chemosensory modalities are processed separately or not. In the hamster, a mammal, Licht and Meredith ('87) found convergence of main and accessory inputs onto single neurons of the vomeronasal amygdala (posteromedial cortical nucleus). An unexpected finding of this work has been the observation of a reciprocal connection between the main and accessory olfactory bulbs. These connections arise from the mitral cells (see Fig. 4D,F) and terminate in the mitral cell layer, inner plexiform layer and part of the granular layer in both olfactory bulbs, providing a direct feedback loop between the MOB and AOB. The nature and meaning of this interrelation is not known, but it strongly suggests that an intimate coordination of the vomeronasal and olfactory systems at this level may be necessary for the performance of complex chemoreception-related tasks. So far, a similar projection (from the MOB to the AOB) has been described only for an amphibian (Kemali and Gugliemotti, '87).
Centrifugal connections of the olfactory bulbs According to our results, the olfactory bulbs are reciprocally connected with the main targets of their centripetal connections. The vomeronasal amygdala, including the nucleus of the accessory olfactory tract, the central and ventromedial amygdaloid nuclei, the nucleus sphericus, and the bed nucleus of the stria terminalis project to the ipsilateral AOB. The contralateral nucleus sphericus (hilus) projects to the AOB through the anterior commissure, and the vomeronasal AON (ventromedial part) projects bilaterally to the AOB. The contralateral fibers course through the stria medullaris-habenular commissure system.
345
The MOB receives bilateral input from the rostral lateral cortex (olfactory cortex) and olfactory AON (dorsomedial part) (the contralateral component coursing through the striae medullaris-habenular commissure system). The reciprocal connections between the olfactory bulbs and their telencephalic target structures may provide feedback loops allowing amplification of the chemosensory inputs. In mammals (Gray et al., '87; Gray and Skinner, '88) epileptiform activity in the MOB elicited by odorant stimuli is mediated and maintained by centrifugal afferents from the olfactory cortex, suggesting that a feedback circuit between both structures is involved. In P. hispanica, these centrifugal projections terminate in the deep (AOB) or superficial (MOB) granular layer. According to Golgi studies (Garcia-Verdugo et al., '87; Llahi and Garcia Verdugo, '891, these zones are occupied by dendritic branches of granular and mitral cells. In addition to these "specific" afferents, both olfactory bulbs share some other sources of afferents in the forebrain: the diagonal band nucleus, the lateral preoptic area, and the caudal dorsal cortex. The projection from the dorsal cortex has been found in Gekko to arise from the rostral third of this cortical area (Hoogland and Vermeulen Van der Zee, '89), whereas our results clearly indicate that it originates in its caudal half. This discrepancy may be attributed to interspecific variance in the organization of the cerebral cortex. The dorsal cortex and the DBN are the targets for nonolfactory aiTerents: the dorsal cortex receives multimodal afferents from the dorsolateral anterior thalamus (Belekhova and Ivazov, '83; Lohman and Van WoerdenVerkley, '78; Bruce and Butler, '84; Martinez-Garcia and Olucha, '88; Martinez-Garcia and Lorente, '90); the DBN is reached by ascending projections from the hypothalamus, raphe nucleus, and other mesencephalic structures (Martinez-Garcia, '88). Hence, it is not likely that these structures participate in processing the chemosensory information; instead they should modulate or modify the olfactory and vomeronasal input depending on the general physiological status of the organism. A similar role for the DBN centrifugal projection has been suggested by Price and Powell ('70) in the rat. Finally, some extratelencephalic nuclei project to both olfactory bulbs, namely, the midbrain raphe nucleus and the locus coeruleus. Gathering together our results and the available data on the serotonergic (Smeets and Steinbusch, '88) and noradrenergic (Smeets and Steinbusch, '89) systems of the Gekko forebrain and midbrain, it can be concluded that the raphe nucleus and locus coeruleus are responsible, respectively, for the serotonergic and noradrenergic innervation of the olfactory bulbs. Our restricted injections in the olfactory bulbs labeled only a few (2-3) neurons in those nuclei. In fact, labeling of locus coeruleus cells was observed only after a relatively large injection into the AOB. However, noradrenergic innervation of the MOB has also been described, suggesting that it actually receives a projection from the locus coeruleus, which may be too diffuse and scarce for being detected by injections of small HRP volumes.
Comparative remarks Our results show that the hodological organization of the olfactory and vomeronasal systems is similar in mammals and lizards, although some differences are observed. The most striking of these is the complete absence in mammals of contralateral olfactory projections (for review, see Swit-
346
zer et al., '85). This feature is parallelled by the absence of commissural connections from telencephalic structures through the stria medullaris-habenular commissure system, which are present in the rest of tetrapoda (Reiner and Karten, '85; Kemali and Gugliemotti, '87). Instead, mammalian olfactory structures projecting contralaterally (AON and pyriform cortex) use the anterior commissure as a route. The MOB in both mammals and reptiles connects reciprocally with the AON (mammals: Broadwell, '75; Scalia and Winans, '75; Dennis and Kerr, '76; Skeen and Hall, '77; De Olmos et al., '78; Shipley and Adamek, '84; De Carlos et al., '89). In mammals, another retrobulbar structure reciprocally connected with the MOB is the subfield tt, of the taenia tecta or precommissural hippocampus, the reptilian counterpart of which may be the dorsomedial part of the AON as defined by us. In fact, the AONm is a rostral extension of the medial cortex (a similar structure in Dipsosaurus was called 'rostral rudiment of the medial cortex' by Ulinski and Peterson, '81) that has been considered as a reptilian homologue of the mammalian hippocampus (Curwen, '37; Goldby and Gamble, '57; Northcutt, '67; Ebbesson and Voneida, '69; Lohman and Mentink, '72). The mammalian olfactory cortex is composed of prepiriform, piriform, and entorhinal areas (Heimer, '69; Scalia and Winans, '75; Skeen and Hall, '77). Its rostral part (prepiriform cortex) projects back bilaterally to the olfactory bulb (Dennis and Kerr, '76; De Olmos et al., '78; De Carlos et al., '89). Likewise, the rostral part of the lacertilian olfactory (lateral) cortex projects bilaterally to the MOB. However, this rostral part of the lateral cortex of lizards projects as well to the 'hippocampal formation' (medial cortex) (Lohman and Mentink, '72; MartinezGarcia et al., '86) like the entorhinal cortex of mammals does (Schwerdtfeger, '84).This fact suggests that, like the visual system (Ebbesson, '811, the olfactory system has undergone a process of parcelation during vertebrate evolution. In mammals (Broadwell and Jacobowitz, '76; Dennis and Kerr, '76; De Olmos et al., '78) and reptiles (results of this work), the main centrifugal afferent of the MOB arises from the DBN. Moreover, some cells of the lacertilian lateral preoptic area caudally adjacent to the DBN project to the MOB; this cell group may be, according to their projections and location, the reptilian counterpart of the mammalian substantia innominata (De Olmos et al., '78). The presence of acetylcholinesterase reactive cells in this position has been recently reported in the tegu lizard (Follett, '89). A projection from the DBN and 'substantia innominata' to the AOB has been found in reptiles (this work) and in some mammals by Broadwell and Jacobowitz ('76). Although these authors interpreted their results as due to spread of injected HRP into the MOB, our results on that point are conclusive, since the MOB and AOB of Podarcis are large enough for restricting injections into either one or the other. The projection of the MOB to superficial structures immersed in the olfactory tract (olfactory tubercle and nucleus of the lateral olfactory tract) is also found in mammals (Broadwell, '75; Scalia and Winans, '75; Skeen and Hall, '77). Concerning the connections of the AOB, the high similarity of the efferent connections in mammals and reptiles has been long discussed by other authors (Halpern, '76; Ulinski and Peterson, '81). Our results support findings of Lohman
F. MARTINEZ-GARCIA ET AL. et al. ('88) according to which there is in reptiles, as in mammals (Broadwell, '75; Skeen and Hall, '771, a direct projection from the AOB to part of the bed nucleus of the stria terminalis. Moreover, the nucleus sphericus, previously compared to the posteromedial cortical nucleus of the mammalian amygdala (Halpern, '76; Ulinski and Peterson, '81) in view of its massive afferent from the AOB, is the origin of a bilateral (with ipsilateral predominance) centrifugal projection to the AOB, like the mammalian posteromedial cortical nucleus of the amygdala (Broadwell and Jacobowitz, '76; De Olmos et al., '78). Likewise, the bed nucleus of the stria terminalis and the bed nucleus of the accessory olfactory tract project to the AOB in reptiles (this work) as well as in mammals (De Olmos et al., '78). The centrifugal connection of the AON to the AOB described in this work has not been observed in mammals (Switzer et al., '85). Our results show that this labeled cell group is a rostral extension of the bed nucleus of the accessory olfactory tract (projecting in mammals to the AOB, De Olmos et al., '78).Hence, this part of the AON may be considered as the rostral tip of the amygdaloid formation, which would reflect the high development that the vomeronasal system has reached in squamate reptiles (Halpern, '87). On the whole, the fiber connections traced in this study hint at many putative homologies of the olfactory system in the brain of reptiles and mammals. However, conclusive data will be obtained only after the investigation of a larger sample of species than those studied so far.
ACKNOWLEDGMENTS This work has been supported in part by a grant from "Accions concertades d'investigacio de 1'Universitat de Valencia" project number 5183.
LITERATURE CITED Belekhova, M.G., and N.I. Ivazov (1983) Analysis of transmission of visual, somatic, and audiovibrational sensory information to the hippocampal cortex of lizards. Neirofiziol. 15:153-159. Bellairs, A. (1969) The Life of Reptiles. London: Weidenfield and Nicholson, 590 pp. Broadwell, R.D. (1975) Olfactory relationships of the telencephalon and diencephalon of the rabbit. I. An autoradiographic study of the efferent connections of the main and accessory olfactory bulbs. J. Comp. Neurol. 163:329-346. Broadwell, R.D., and D.M. Jacobowitz (1976) Olfactory relationships of the telencephalon and diencephalon in the rabbit. 111. The ipsilateral centrifugal fibers to the olfactory bulbar and retrobulbar formations. J. Comp. Neurol. 170:321-346. Bruce, L., and A.B. Butler (1984) Telencephalic connections in lizards. I. Projections to cortex. J. Cornp. Neurol. 229.585-601. Burghart, G. (1970) Chemical perception in reptiles. In J.W. Johnston, D.G. Moulton and A. Turk (eds): Communication by Chemical Signals. New York: Appleton-Century-Crofts, pp. 24 1-308. Curwen, A.O. (1937) The telencephalon of Tupinambis nigropunctatus. I. Medial and cortical areas. J. Comp. Neurol. 02375-404. Curwen, A.O. (1939) The telencephalon of Tupinanbis nigropunctatus. 111. Arnygdala. J. Comp. Neurol. 713313-636. De Carlos, J.A., L. Lopez-Mascaraque, and F. Valverde (1989) Connections of the olfactory bulb and nucleus olfactorius anterior in the hedgehog (Erinaceus europaeus): Fluorescent tracers and HRP study. J. Comp. Neurol. 279:601-618. De Olmos, J.S., H. Hardy, and L. Heirner (1978) The afferent connections of the main and accessory bulb formations in the rat: An experimental HRP study. J. Cornp. Neurol. 181r213-244. Dennis, B.J., and D.B. Kerr (1976) Origin of the olfactory bulb centrifugal fibers in the cat. Brain Res. 110:593-600.
LIZARDOLFACTORYBULB CONNECTIONS Ebbesson, S.O.E. (1981) Evolution and ontogeny of neural circuits. Behav. Brain Sci. 7,321-366. Ebbesson, S.O.E., and T.J. Voneida (1969) The cytoarchitecture of the tegu lizard Tupinambis nigropunctatus. Brain Behav. Evol. 2:431466. Follett, K.A. (1989) A telencephalospinal projection in the tegu lizard (Tupinambrs teguixin). Brain Res. 496:89-97. Gabe, M., and H. St. Girons (1976) Contribution a la morphologie comparee des fosses nasales et de leurs annexes chez les lepidosauriens. Mem. Mus. Nat. D‘Hist. Nat. Ser. A. 98:l-87. Gamble, H.J. (1952) An experimental study on the secondary olfactory connections in Lacerta uiridis. J. Anat. 86:180-196. Garcia-Verdugo, J.M., S. Llahi, I. Farinas, and V. Martin (1987) Laminar organization of the main olfactory bulb ofPodarcis hispanica: an electron microscopic and Golgi study. J. Hirnforsch. 27:87-100. Goldby, R., and H.J. Gamble (1957)The reptilian cerebral hemispheres. Biol. Rev. 32,383420. Gray, C.M., and J.E. Skinner (1988) Centrifugal regulation of neuronal activity in the olfactory bulb of the waking rabbit as revealed by reversible cryogenic blockade. Exp. Brain Res. 69:378-386. Gray, C.M., W.J. Freeman, and J.E. Skinner (1987) Induction and maintenance of epileptiform activity in the rabbit olfactory bulb depends on centrifugal input. Exp. Brain Res. 68:210-212. Halpern, M. (1976) The efferent connections of the olfactory bulb and accessory olfactory bulb in the snakes Thamnophis sirtalis and Thamnophis radix. J. Morphol. 150:553-578. Halpern, M. (1980) The telencephalon of snakes. In S.O.E. Ebbesson (ed): Comparative Neurology of the Telencephalon. New York: Plenum Press, pp. 257-294. Halpern, M. (1987) The organization and function of the vomeronasal system. Ann. Rev. Neurosci. lOt325-362. Heimer, L. (1969)The secondary olfactory connections in mammals, reptiles and sharks. Ann. N.Y. Acad. Sci. 16729-146. Hoogland, P.V., and E. Vermeulen-Van der Zee (1989) Efferent connections of the dorsal cortex of the lizard Gekko gecko studied with Phaseolus vulgaris-leucoagglutinin. J. Comp. Neurol. 285289-303. Kemali, M., andV. Gugliemotti (1987) A horseradish peroxidase study of the olfactory system of the frog, Rana esculenta. J. Comp. Neurol. 263:400417. Licht, G., and M. Meredith (1987) Convergence of main and accessory olfactory pathways onto single neurons in the hamster amygdala. Exp. Brain Res. 69:7-18. Llahi, S., and J.M. Garcia-Verdugo (1989) Neuronal organization of the accessory olfactory bulb of the lizard Podarcis hispanica: Golgi study. J. Morphol. 202:13-28. Lohman, A.H.M., and I. Van Woerden-Verkley (1978) Ascending connections to the forebrain in the tegu lizard. J. Comp. Neurol. 182:555-594. Lohman, A.H.M., and G.M. Mentink (1972) Some cortical connections of the tegu lizard (Tupinambisteguixin). Brain Res. 45:325-344. Lohman, A.H.M., P.V. Hoogland, and R.J.G.M. Witjes (1988) Projections from the main and accessory olfactory bulbs to the amygdaloid complex in the lizard Gekko gecko. In W.K. Schwerdtfeger and W.J.A.J. Smeets (eds): The Forebrain of Reptiles. Current Concepts of Structure and Function. Base1 Karger, pp. 41-49. Martinez-Garcia, F. (1988) Conexiones de las ireas corticales y subcorticales del telencefalo de la lagartija comdn Podarcis hispanica. Doctoral dissertation, University of Valencia.
347 Martinez-Garcia, F., and M.J. Lorente (1988) Fibre connections of the amygdala in the lizard, Podarcis hispanica. 11th Meeting of the E.N.A., Satellite Symposium, “Comparative aspects on the structure and development of the forebrain in lower vertebrates.” Zurich, Switzerland. Martinez-Garcia, F., and M.J. Lorente (1990) Thalamo-cortical projections in the lizard Podarcis hispanica. In W.K. Schwerdtfeger and P. Germroth (eds): The Forebrain in nonmammals. New Aspects of Structure and Development. Berlin: Springer-Verlagpp. 93-102. Martinez-Garcia, F., and F.E. Olucha (1988) Afferent projections to the Timm-positive cortical areas of the telencephalon of lizards. In W.K. Schwerdtfeger and W.J.A.J. Smeets (eds): The Forebrain of Reptiles. Current Concepts of Structure and Function. Base1 Karger, pp. 30-40. Martinez-Garcia, F., M. Amiguet, F. Olucha, and C. L6pez-Garcia (1986) Connections of the lateral cortex in the lizard Podarcis hispanica. Neurosci. Lett. 63:39-44. Northcutt, R.G. (1967)Architectonic studies of the telencephalon of Iguana iguana.J. Comp. Neurol. 130:109-148. Olucha, F., F. Martinez-Garcia, and C. L6pez-Garcia (1985) A new stabilizing agent for the tetramethyl benzidine (TMB) reaction product in the histochemical detection of horseradish peroxidase (HRP). 3. Neurosci. Meth. 13t131-138. Price, J.L., and P.J. Powell (1970) An experimental study of the origin and the course of the centrifugal fibers to the olfactory bulb in the rat. J. Anat. 107t215-237. Raisman, G. 11972)An experimental study of the projection of the amygdala to the accessory olfactory bulb and its relationship to the concept of a dual olfactory system. Exp. Brain Res. 14:395408. Reiner, A,, and H.G. Karten (1985) Comparison of the olfactory bulb projections in pigeon and turtles. Brain Behav. Evol. 27t11-27. Scalia, F., M. Halpern, and W. Riss (1969) Olfactory bulb projections in the South American caiman. Brain Bebav. Evol. 2:238-262. Scalia, F., and S.S. Winans (1975) The differential projections of the olfactory bulb and accessory olfactory bulb in mammals. Brain Res. 161:31-56. Scbwerdtfeger, W.K. (1984) Structure and Fiber Connections of the Hippocampus: A Comparative Study. Berlin: Springer-Verlag, 74 pp. Shipley, M.T., and G.D. Adamek (1984) The connections of the mouse olfactory bulb: a study using orthograde and retrograde transport of wheatgerm agglutinin conjugated to horseradish peroxidase. Brain Res. Bull. 12:669-688. Skeen, L.C., and W.C. Hall (1977) Efferent projections of the main and the accessory olfactory bulb in tree shrew (Tupaia glis). J. Comp. Neurol. 172.1-36. Smeets, W.J.A.J., and H.W.M. Steinbusch (1988) Distribution of serotonin immunoreactivity in the forebrain and midbrain of the lizard Gekko gecko. J. Comp. Neurol. 271:419-434. Smeets, W.J.A.J., and H.W.M. Steinbusch (1989)Distribution of noradrenaline immunoreactivity in the forebrain and midbrain of the lizard Gekko gecko. J. Comp. Neurol. 285t453-466. Switzer, R.C., J. De Olmos, and L. Heimer (1985) Olfactory system. In G. Paxinos (ed): The Rat Nervous System. Sydney: Academic Press, pp. 1-36. Ulinski, P.S. (1983) Dorsal ventricular ridge: A Treatise on Forebrain Organization in Reptiles and Birds. New York: John Wiley & Sons. Ulinski, P.S., and E.H. Peterson (1981) Patterns of olfactory projections in the desert iguana, Dipsosaurus dorsalis. J. Morphol. 168:189-227.
