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A reassessment of the direct projections of the olfactory bulb
Brain Research, 151 (1978) 375-380 © Elsevier/North-Holland Biomedical Press
375
A reassessment of the direct projections of the olfactory bull,b
BLAIR H. T U R N E R and MORTIMER MISHKIN Department of Anatomy, Howard University Medical School, Washington, D.C. 20059 and Laboratory of Neuropsychology, National Institute of Mental Health, Bethesda, Md. 20014 (U.S..4.)
(Accepted February 16th, 1978)
Recent experiments in a variety of mammalian species have shown that the projections of the olfactory bulb are exclusively ipsilateral and that the projections to all the ipsilateral target areas are restricted to cortical layer IA1, 6-9,tl. The present report is a reassessment of some findings that appear to diverge from the foregoing scheme. In the course of a study on the terminal distribution of the olfactory bulb of the rhesus monkey (Macaca mulatta)9, olfactory projections that varied from the pattern outlined above were noticed in a brain processed by the Fink-Heimer technique 2 following a 10-day survival period, and in a brain processed by the autoradiographic technique following a 12-day survival. In the first case, the aberrant degeneration was very light and therefore subject to the interpretation that it consisted of insufficiently suppressed normal fibers. In the autoradiographic case, however, it was clear that there were olfactory bulb projections to the deeper layers of certain ipsilateral olfactory areas, to the supraoptic crest (one of the circumventricular organs) at the midline, and even to some contralateral olfactory areas. Ipsilateral terminations deeper than layer IA have been explicitly denied in autoradiographic material 6 and, as a result, the deeper projections that have often been seen with the experimental silver methods 3,7,1t have been attributed to transneuronal degeneration secondary to damage of the olfactory bulb 6. As for midline or contralateral connections from the olfactory bulb, there has been no suggestion of these with any of the newer techniques. Furthermore, our own autoradiographic and Fink-Heimer material in cases with survival times ranging from one to 4 days also revealed none of these ipsilateral or contralateral variations from the olfactory distribution pattern established in other species. Thus, the weight of the evidence led us to assume that in the animal that survived 10 days the discrepant findings either were artifactual or, as noted above, represented transneuronal degeneration, while in the animal that survived 12 days the anomalous projections were due to transneuronal uptake of labelled protein that had been released postsynaptically. On the supposition that such transneuronal uptake would be seen even more clearly with a still longer survival period, the olfactory bulb of another monkey was injected and its brain processed with the autoradiographic method after a 20-day survival. In this animal, the ipsilateral olfactory afferents to laye]s IB, II and III of the olfactory tubercle and frontal prepiriform cortex were confirmed, and a faint projection
376 TABLE I Effects o f survival time on the appearance o f ol['actory bulb projections Olfactory structure
Olfactory bulb Olfactory tract Anterior olfactory nucleus Olfactory tubercle Frontal prepiriform cortex Temporal prepiriform cortex Periamygdaloid cortex Medial amygdaloid nucleus Entorhinal cortex Anterior commissure Supraoptic crest
Short survival (4 days or less)
Long survival (10 days or more)
Ipsilateral
Contralateral
lpsilateral
Contralateral *
injection site present layer IA layer IA layer IA layer IA layer 1A layer IA layer IA absent absent
absent absent absent absent absent absent absent absent absent absent absent
injection site present layer IA layers I-Ili layers I-I11 layer IA layer IA layer IA layer IA present present
all layers present absent layer IA layer IA layer IA absent absent absent present present
* Not present in animal surviving 20 days. was seen crossing the anterior commissure and continuing in the fascicles of the contralateral anterior limb of the commissure. This labelling soon disappeared, however, and there was no evidence of terminations in the contralateral olfactory bulb or cortex, or in the supraoptic crest. Since the findings after a 20-day survival only partially confirmed the 10-day Fink-Heimer and 12-day autoradiographic results, it was presumed that the anomalous contralateral projections required an optimal time for their appearance. However, it still seemed best to interpret such projections as transneuronal effects, and they were therefore excluded from our earlier description of the direct olfactory bulb connections in the rhesus monkey 9. Upon reconsideration, however, we feel that the transneuronal hypothesis does not adequately explain the discrepant findings, and that the alternative possibility must be considered that all the projections we found, not just those to cortical layer IA on the ipsilateral side, are in fact direct projections from the olfactory bulb. Our reasons for advancing this proposal will be given following a more detailed presentation of the experimental data. The experimental animals were 6 juvenile male monkeys. In 5 of these, 1-1.5 bd of [aH]proline or leucine were injected into the left olfactory bulb, the animals allowed to survive 18 h, or 2, 4, 12 or 20 days, and their brains prepared for autoradiography. In the 2-day survival case, some damage occurred to the bulb, and this brain was also prepared by the Fink-Heimer technique. The left olfactory tract was cut in a sixth monkey, and this animal was allowed to survive 10 days. Its brain was stained by the Fink-Heimer method. The details of the surgical and histological procedures were presented in the earlier report 9. A comparison of the olfactory bulb connections seen after short (4 days or less) and long (10 days or more) survival times is presented in Table I. Following a short survival period, both the Fink-Heimer and autoradiographic methods show a terminal distribution pattern that coincides with that observed in other species: the projections,
Fig. 1. A: a fiber-stainedt° section indicating the lamination of the olfactory tubercle and frontal prepiriform cortex lying between the olfactory tract (OT) below and the putamen (PUT) above. In this figure, and in B and C, tissue to the left of the lines demarcating the cortical layers (IA, IB, II and III) is olfactory tubercle, while tissue to the right of these lines is frontal prepiriform cortex. The level shown here, and in B and C, is approximately 22 mm anterior to the interaural line. 32 x .B : an autoradiograph from an animal that survived 18 h. Note the sharp boundary between layers IA and IB, and the absence of a projection to the cortical layers deeper than IA. Autoradiographs of brains in which the survival time was 4 days or less (as in this animal) did not display the density of silver grains of those in which the survival time was longer (as in the animal illustrated below). 32 x . C: an autoradiograph from an animal that survived 12 days taken from the same brain shown in A. Note the group of fascicles that have detached from the olfactory tract to fan out to the deeper cortical layers. Labelling is heaviest in layer IA, but it is clearly present in all layers. The density of the silver grains was greatest in this animal and somewhat less in the animal that survived 20 days, although in the latter case the density was still greater than that observed when survival was 4 days or less. 32 x .
378
Fig. 2. Two structures that receive projections from the olfactory bulb by way of the anterior commissure seen in the animal that survived 12 days. A : a projection to layer IA of the contralateral temporal prepiriform cortex. Note the sharp boundary demarcating layers 1A and 1B. This projection was invisible at the magnification of Fig. 1. The section is taken from approximately 20 mm anterior to the interaural line. 130 x. B: a projection to the supraoptic crest, i.e. organum vasculosum lamina terminalis. This section is taken at a level that is approximately 17 mm anterior to the interaural line. The supraoptic crest lies in the most anterior tip of the third ventricle, immediately above the most anterior part of the optic chiasm. 130 x. coursing through the olfactory tract, end in layer IA of the 7 ipsilateral target areas listed in the table. Projections to two o f these ipsilateral target areas, the olfactory tubercle and the frontal prepiriform cortex, are shown in Fig. 1B. When the survival period is longer, however, the ipsilateral afferents to the olfactory tubercle and the frontal division ofprepiriform cortex ends in all 3 cortical layers, as shown in Fig. 1C. Furthermore, there is a projection via the anterior commissure to layer IA of the contralateral olfactory tubercle, and frontal and temporal prepiriform cortices; the last of these is illustrated in Fig. 2A. Finally, as shown in Fig. 2B, another projection via the anterior commissure is seen to a structure not previously suspected o f receiving olfactory connections, the supraoptic crest. In reconsidering whether these anomalous olfactory terminations could reflect transneuronal processes, we may assume that the anterior olfactory nucleus is the most likely site for either passive spread or transneuronal uptake of the radioactively labelled material (autoradiographic method) or for transneuronal cellular degeneration ( F i n k Heimer method). The direct argument against passive spread is that careful examination of the autoradiographs revealed no evidence of extraneuronal labelling along the approximately 12 m m o f the olfactory tract that separates the olfactory bulb from the anterior olfactory nucleus; rather, all o f the injected material appeared to be confined to the olfactory bulb itself. But there is also a compelling indirect argument against passive
379 spread, and this argument applies equally well against the possibility that transneuronal effects had occurred within the anterior olfactory nucleus. Broadwell 1 has shown in the rabbit that the anterior olfactory nucleus projects not to layer IA of the contralateral prepiriform cortex, but to layers IB, II and III of this cortex. It our contralateral projection were the result of transneuronal uptake or degeneration, then terminals should have been absent in layer IA, and present in layers IB, II and III. That just the opposite occurred is demonstrated in Fig. 2A. The same considerations probably argue against transneuronal effects occurring in other ipsilateral olfactory areas as well, since what little information is available suggests that they too distribute only to the deeper layers contralaterallyl,6. Since the terminal distribution to the contralateral prepiriform cortices did involve layer IA selectively, and since this is the layer that is widely believed to be the sole recipient ofipsilateral afferents from the olfactory bulb, a question is raised as to whether the so-called contralateral projection in our cases may actually have represented an ipsilateral projection following spread of the injected material to the opposite bulb (autoradiographic method) or inadvertent damage to this bulb (Fink-Heimer method). Such an interpretation, however, fails to account either for the presence of labelled fibers in the anterior commissure or for the absence of terminations in the well-established ipsilateral target areas outside the prepiriform cortices, namely, the periamygdaloid cortex, the medial amygdaloid nucleus and the entorhinal cortex. In summary, our reevaluation of the experimental findings seems to have eliminated the obvious alternatives to the interpretation that the projections listed in Table I are direct terminals of the olfactory bulb. Such a conclusion, at least with respect to the deep ipsilateral afferents, is not new. Discovered originally by White 11, using the Nauta technique, and corroborated by both Heimer 3 and Price and Powell 7 using the newer versions of the silver impregnation technique, the finding of olfactory bulb projections to the deeper layers of olfactory cortex was denied as being direct only when such terminals did not appear in autoradiographic material 0. The autoradiographic evidence presented here, however, suggests that the original findings with the degeneration methods may have been valid after all. The conclusion must still remain tentative, because the deep ipsilateral projections were seen in the autoradiographs only after relatively long survival times, and it remains unclear why this was so. Unlike the evidence for deep ipsilateral projections, evidence that direct efferents of the bulb cross in the anterior commissure is new. These must be regarded even more tentatively, since they seemed to require not just a long but rather an optimal survival time for their appearance, and again the explanation for such a phenomenon is unclear. Although we advance it hesitantly, the suggestion that the olfactory bulb projects directly to the contralateral olfactory cortex and to the supraoptic crest, an endocrine organ 5 containing luteinizing hormone-releasing hormone 4, is sufficiently provocative to merit further study. We wish to thank Margaret E. Knapp for her skillful technical assistance. This research was supported in part by N I M H Grant MH25495.
380 1 Broadwell, R. D., Olfactory relationships of the telencephalon and diencepbalon in the rabbit II. An autoradiographic and horseradish peroxidase study of the efferent connections of the anterior olfactory nucleus, J. comp. NeuroL, 164 (1975) 389-410. 2 Fink, R. and Heimer, L., Two methods for selective silver impregnation of degenerating axons and their synaptic endings in the central nervous system, Brain Research, 4 (1967) 369-374. 3 Heimer, L., Synaptic distribution of centripetal and centrifugal nerve fibers in the olfactory system of the rat. An experimental anatomical study, J. Anat. (Lond.), 103 (1968) 413-432. 4 Kizer, J. S., Palkovits, M. and Brownstein, M. J., Releasing factors in the circumventricular organs of the rat brain, Endocrinology, 98 (1976) 311-317. 5 Le Beux, Y. J., An ultrastructural study of the neurosecretory cells of the medial vascular prechiasmatic gland, Z. Zellforsch., 127 (1972) 439-461. 6 Price, J. L., An autoradiographic study of complementary laminar patterns of termination of afferent fibers to the olfactory cortex, J. comp. Neurol., 150 (1973) 87-108. 7 Price, J. L. and Powell, T. P. S., Certain observations on the olfactory pathway, J. A nat. (Lond.), 110 (1971) 105-126. 8 Scalia, F. and Winans, S. S., The differential projections of the olfactory bulb and accessory olfactory bulb in mammals, J. comp. Neurol., 161 (1975) 31-56. 9 Turner, B. H., Gupta, K. C. and Mishkin, M., The locus and cytoarchitecture of the olfactory bulb projection areas in Macaca mulatta, J. comp. Neurol., 177 (1978) 381-396. 10 Vogt, B., A reduced silver stain for normal axons in the central nervous system, Physiol. Behav., 13 (1974) 837-840. 11 White, L. E., Jr., Olfactory bulb projections of the rat, Anat. Rec., 152 (1965) 465-480.
375
A reassessment of the direct projections of the olfactory bull,b
BLAIR H. T U R N E R and MORTIMER MISHKIN Department of Anatomy, Howard University Medical School, Washington, D.C. 20059 and Laboratory of Neuropsychology, National Institute of Mental Health, Bethesda, Md. 20014 (U.S..4.)
(Accepted February 16th, 1978)
Recent experiments in a variety of mammalian species have shown that the projections of the olfactory bulb are exclusively ipsilateral and that the projections to all the ipsilateral target areas are restricted to cortical layer IA1, 6-9,tl. The present report is a reassessment of some findings that appear to diverge from the foregoing scheme. In the course of a study on the terminal distribution of the olfactory bulb of the rhesus monkey (Macaca mulatta)9, olfactory projections that varied from the pattern outlined above were noticed in a brain processed by the Fink-Heimer technique 2 following a 10-day survival period, and in a brain processed by the autoradiographic technique following a 12-day survival. In the first case, the aberrant degeneration was very light and therefore subject to the interpretation that it consisted of insufficiently suppressed normal fibers. In the autoradiographic case, however, it was clear that there were olfactory bulb projections to the deeper layers of certain ipsilateral olfactory areas, to the supraoptic crest (one of the circumventricular organs) at the midline, and even to some contralateral olfactory areas. Ipsilateral terminations deeper than layer IA have been explicitly denied in autoradiographic material 6 and, as a result, the deeper projections that have often been seen with the experimental silver methods 3,7,1t have been attributed to transneuronal degeneration secondary to damage of the olfactory bulb 6. As for midline or contralateral connections from the olfactory bulb, there has been no suggestion of these with any of the newer techniques. Furthermore, our own autoradiographic and Fink-Heimer material in cases with survival times ranging from one to 4 days also revealed none of these ipsilateral or contralateral variations from the olfactory distribution pattern established in other species. Thus, the weight of the evidence led us to assume that in the animal that survived 10 days the discrepant findings either were artifactual or, as noted above, represented transneuronal degeneration, while in the animal that survived 12 days the anomalous projections were due to transneuronal uptake of labelled protein that had been released postsynaptically. On the supposition that such transneuronal uptake would be seen even more clearly with a still longer survival period, the olfactory bulb of another monkey was injected and its brain processed with the autoradiographic method after a 20-day survival. In this animal, the ipsilateral olfactory afferents to laye]s IB, II and III of the olfactory tubercle and frontal prepiriform cortex were confirmed, and a faint projection
376 TABLE I Effects o f survival time on the appearance o f ol['actory bulb projections Olfactory structure
Olfactory bulb Olfactory tract Anterior olfactory nucleus Olfactory tubercle Frontal prepiriform cortex Temporal prepiriform cortex Periamygdaloid cortex Medial amygdaloid nucleus Entorhinal cortex Anterior commissure Supraoptic crest
Short survival (4 days or less)
Long survival (10 days or more)
Ipsilateral
Contralateral
lpsilateral
Contralateral *
injection site present layer IA layer IA layer IA layer IA layer 1A layer IA layer IA absent absent
absent absent absent absent absent absent absent absent absent absent absent
injection site present layer IA layers I-Ili layers I-I11 layer IA layer IA layer IA layer IA present present
all layers present absent layer IA layer IA layer IA absent absent absent present present
* Not present in animal surviving 20 days. was seen crossing the anterior commissure and continuing in the fascicles of the contralateral anterior limb of the commissure. This labelling soon disappeared, however, and there was no evidence of terminations in the contralateral olfactory bulb or cortex, or in the supraoptic crest. Since the findings after a 20-day survival only partially confirmed the 10-day Fink-Heimer and 12-day autoradiographic results, it was presumed that the anomalous contralateral projections required an optimal time for their appearance. However, it still seemed best to interpret such projections as transneuronal effects, and they were therefore excluded from our earlier description of the direct olfactory bulb connections in the rhesus monkey 9. Upon reconsideration, however, we feel that the transneuronal hypothesis does not adequately explain the discrepant findings, and that the alternative possibility must be considered that all the projections we found, not just those to cortical layer IA on the ipsilateral side, are in fact direct projections from the olfactory bulb. Our reasons for advancing this proposal will be given following a more detailed presentation of the experimental data. The experimental animals were 6 juvenile male monkeys. In 5 of these, 1-1.5 bd of [aH]proline or leucine were injected into the left olfactory bulb, the animals allowed to survive 18 h, or 2, 4, 12 or 20 days, and their brains prepared for autoradiography. In the 2-day survival case, some damage occurred to the bulb, and this brain was also prepared by the Fink-Heimer technique. The left olfactory tract was cut in a sixth monkey, and this animal was allowed to survive 10 days. Its brain was stained by the Fink-Heimer method. The details of the surgical and histological procedures were presented in the earlier report 9. A comparison of the olfactory bulb connections seen after short (4 days or less) and long (10 days or more) survival times is presented in Table I. Following a short survival period, both the Fink-Heimer and autoradiographic methods show a terminal distribution pattern that coincides with that observed in other species: the projections,
Fig. 1. A: a fiber-stainedt° section indicating the lamination of the olfactory tubercle and frontal prepiriform cortex lying between the olfactory tract (OT) below and the putamen (PUT) above. In this figure, and in B and C, tissue to the left of the lines demarcating the cortical layers (IA, IB, II and III) is olfactory tubercle, while tissue to the right of these lines is frontal prepiriform cortex. The level shown here, and in B and C, is approximately 22 mm anterior to the interaural line. 32 x .B : an autoradiograph from an animal that survived 18 h. Note the sharp boundary between layers IA and IB, and the absence of a projection to the cortical layers deeper than IA. Autoradiographs of brains in which the survival time was 4 days or less (as in this animal) did not display the density of silver grains of those in which the survival time was longer (as in the animal illustrated below). 32 x . C: an autoradiograph from an animal that survived 12 days taken from the same brain shown in A. Note the group of fascicles that have detached from the olfactory tract to fan out to the deeper cortical layers. Labelling is heaviest in layer IA, but it is clearly present in all layers. The density of the silver grains was greatest in this animal and somewhat less in the animal that survived 20 days, although in the latter case the density was still greater than that observed when survival was 4 days or less. 32 x .
378
Fig. 2. Two structures that receive projections from the olfactory bulb by way of the anterior commissure seen in the animal that survived 12 days. A : a projection to layer IA of the contralateral temporal prepiriform cortex. Note the sharp boundary demarcating layers 1A and 1B. This projection was invisible at the magnification of Fig. 1. The section is taken from approximately 20 mm anterior to the interaural line. 130 x. B: a projection to the supraoptic crest, i.e. organum vasculosum lamina terminalis. This section is taken at a level that is approximately 17 mm anterior to the interaural line. The supraoptic crest lies in the most anterior tip of the third ventricle, immediately above the most anterior part of the optic chiasm. 130 x. coursing through the olfactory tract, end in layer IA of the 7 ipsilateral target areas listed in the table. Projections to two o f these ipsilateral target areas, the olfactory tubercle and the frontal prepiriform cortex, are shown in Fig. 1B. When the survival period is longer, however, the ipsilateral afferents to the olfactory tubercle and the frontal division ofprepiriform cortex ends in all 3 cortical layers, as shown in Fig. 1C. Furthermore, there is a projection via the anterior commissure to layer IA of the contralateral olfactory tubercle, and frontal and temporal prepiriform cortices; the last of these is illustrated in Fig. 2A. Finally, as shown in Fig. 2B, another projection via the anterior commissure is seen to a structure not previously suspected o f receiving olfactory connections, the supraoptic crest. In reconsidering whether these anomalous olfactory terminations could reflect transneuronal processes, we may assume that the anterior olfactory nucleus is the most likely site for either passive spread or transneuronal uptake of the radioactively labelled material (autoradiographic method) or for transneuronal cellular degeneration ( F i n k Heimer method). The direct argument against passive spread is that careful examination of the autoradiographs revealed no evidence of extraneuronal labelling along the approximately 12 m m o f the olfactory tract that separates the olfactory bulb from the anterior olfactory nucleus; rather, all o f the injected material appeared to be confined to the olfactory bulb itself. But there is also a compelling indirect argument against passive
379 spread, and this argument applies equally well against the possibility that transneuronal effects had occurred within the anterior olfactory nucleus. Broadwell 1 has shown in the rabbit that the anterior olfactory nucleus projects not to layer IA of the contralateral prepiriform cortex, but to layers IB, II and III of this cortex. It our contralateral projection were the result of transneuronal uptake or degeneration, then terminals should have been absent in layer IA, and present in layers IB, II and III. That just the opposite occurred is demonstrated in Fig. 2A. The same considerations probably argue against transneuronal effects occurring in other ipsilateral olfactory areas as well, since what little information is available suggests that they too distribute only to the deeper layers contralaterallyl,6. Since the terminal distribution to the contralateral prepiriform cortices did involve layer IA selectively, and since this is the layer that is widely believed to be the sole recipient ofipsilateral afferents from the olfactory bulb, a question is raised as to whether the so-called contralateral projection in our cases may actually have represented an ipsilateral projection following spread of the injected material to the opposite bulb (autoradiographic method) or inadvertent damage to this bulb (Fink-Heimer method). Such an interpretation, however, fails to account either for the presence of labelled fibers in the anterior commissure or for the absence of terminations in the well-established ipsilateral target areas outside the prepiriform cortices, namely, the periamygdaloid cortex, the medial amygdaloid nucleus and the entorhinal cortex. In summary, our reevaluation of the experimental findings seems to have eliminated the obvious alternatives to the interpretation that the projections listed in Table I are direct terminals of the olfactory bulb. Such a conclusion, at least with respect to the deep ipsilateral afferents, is not new. Discovered originally by White 11, using the Nauta technique, and corroborated by both Heimer 3 and Price and Powell 7 using the newer versions of the silver impregnation technique, the finding of olfactory bulb projections to the deeper layers of olfactory cortex was denied as being direct only when such terminals did not appear in autoradiographic material 0. The autoradiographic evidence presented here, however, suggests that the original findings with the degeneration methods may have been valid after all. The conclusion must still remain tentative, because the deep ipsilateral projections were seen in the autoradiographs only after relatively long survival times, and it remains unclear why this was so. Unlike the evidence for deep ipsilateral projections, evidence that direct efferents of the bulb cross in the anterior commissure is new. These must be regarded even more tentatively, since they seemed to require not just a long but rather an optimal survival time for their appearance, and again the explanation for such a phenomenon is unclear. Although we advance it hesitantly, the suggestion that the olfactory bulb projects directly to the contralateral olfactory cortex and to the supraoptic crest, an endocrine organ 5 containing luteinizing hormone-releasing hormone 4, is sufficiently provocative to merit further study. We wish to thank Margaret E. Knapp for her skillful technical assistance. This research was supported in part by N I M H Grant MH25495.
380 1 Broadwell, R. D., Olfactory relationships of the telencephalon and diencepbalon in the rabbit II. An autoradiographic and horseradish peroxidase study of the efferent connections of the anterior olfactory nucleus, J. comp. NeuroL, 164 (1975) 389-410. 2 Fink, R. and Heimer, L., Two methods for selective silver impregnation of degenerating axons and their synaptic endings in the central nervous system, Brain Research, 4 (1967) 369-374. 3 Heimer, L., Synaptic distribution of centripetal and centrifugal nerve fibers in the olfactory system of the rat. An experimental anatomical study, J. Anat. (Lond.), 103 (1968) 413-432. 4 Kizer, J. S., Palkovits, M. and Brownstein, M. J., Releasing factors in the circumventricular organs of the rat brain, Endocrinology, 98 (1976) 311-317. 5 Le Beux, Y. J., An ultrastructural study of the neurosecretory cells of the medial vascular prechiasmatic gland, Z. Zellforsch., 127 (1972) 439-461. 6 Price, J. L., An autoradiographic study of complementary laminar patterns of termination of afferent fibers to the olfactory cortex, J. comp. Neurol., 150 (1973) 87-108. 7 Price, J. L. and Powell, T. P. S., Certain observations on the olfactory pathway, J. A nat. (Lond.), 110 (1971) 105-126. 8 Scalia, F. and Winans, S. S., The differential projections of the olfactory bulb and accessory olfactory bulb in mammals, J. comp. Neurol., 161 (1975) 31-56. 9 Turner, B. H., Gupta, K. C. and Mishkin, M., The locus and cytoarchitecture of the olfactory bulb projection areas in Macaca mulatta, J. comp. Neurol., 177 (1978) 381-396. 10 Vogt, B., A reduced silver stain for normal axons in the central nervous system, Physiol. Behav., 13 (1974) 837-840. 11 White, L. E., Jr., Olfactory bulb projections of the rat, Anat. Rec., 152 (1965) 465-480.