Forebrain Projections of the Pigeon Olfactory Bulb GARL KALMAN RIEKE * AND BERNICE M. WENZEL Department of Physiology and the Brain Research Institute Center for the Health Sciences, University of California at Los Angeles, Los Angeles, California 90024

ABSTRACT The olfactory system of the pigeon (Columba livia) was examined. Our electrophysiological and experimental neuroanatomical (Fink-Heimer technique) data showed that axons from the olfactory bulb terminated in both sides of the forebrain. The cortex prepiriformis (olfactory cortex), the hyperstriatum ventrale and the lobus parolfactorius comprised the uncrossed terminal field. The crossed field included the paleostriatum primitivum and the caudal portion of the lobus parolfactorius, areas which were reached through the anterior commissure. In this report the relationships between areas that receive olfactory information and the possible roles that olfaction plays in the birds' behavior are discussed. The idea that olfaction is a functional sensory system in a number of avian species has gained support slowly. The evidence for this conclusion comes from three disciplines: (1) ethological studies (Stager, '64; Wenzel, '68, '71; Papi et al., '71, '72; Grubb, '74; Rausch et al., ' 7 9 , (2) controlled behavioral studies using odor discrimination or odor cues (Michelsen,'59; Henton et al., '66; Wenzel and Salzman, '68; Wenzel and Sieck, '72; Shallenberger, '75) and (3) electrophysiological studies (Tucker, '65; Shibuya and Tucker, '67; Sieck and Wenzel, '69; Macadar et al., '75; Rieke and Wenzel, '75). In the ethological study of Wenzel ('68) the kiwi (Apteryx australisi was shown to use olfaction in its curious food seeking behavior. The turkey vulture (Cathartes aura) is a scavenger and as Stager's ('64) study has shown it can locate carrion on the basis of olfactory cues alone. In the breeding season the Leach's petrel (Oceanodroma leucorhoa) finds its burrow and recognizes its chicks by means of olfactory cues (Grubb, '74). The exact role of the olfactory system in the homing behavior of pigeons is not understood. However, Papi et al. ('71, '72) have shown that experienced homing pigeons do not return to the home loft successfully after their olfactory nerves have been transected. Pigeons have been used in a variety of controlled behavioral studies. Michelsen ('59) has reported that pigeons can be trained to discriminate between the presence or absence of J. MORPH. (1978) 158: 41-56

a test odor and to respond accordingly. Wenzel and Sieck ('72) have reported that the heart and respiratory rates of the pigeon changed significantly upon presentation of odorants. Electrophysiological studies have provided the strongest evidence for functional olfactory receptor neurons. Tucker ('65) clearly established that many birds have in their olfactory mucosa receptor neurons that respond to odorants. Shibuya and Tucker ('67) introduced odorants into the nasal cavity of the black and turkey vultures and recorded extracellularly the unit activity of the olfactory receptor neurons in the bird's mucosa. Sieck and Wenzel ('69) demonstrated that neurons of the pigeon bulb responded to test odorants. Transection of the olfactory nerves abolished the normally observed odor-induced desynchronization and spindling of the EEG activi t y from the olfactory bulbs in the test birds. The most recent electrophysiological studies of the pigeon olfactory system have demonstrated that the olfactory bulb is connected to a number of forebrain centers (Rieke and Wenzel, '75; Macadar et al., '75; Hutchison et al., '77). Apparently olfaction is a functional sensory system a t least in some avian species; however, very little is known concerning the cen' This research was supported by NIH Grant NS-10353 to B. M Wenzel and a postdoctoral traineeshlp to G. K. Rieke through Training Grant MH-06415 to the Brain Research Institute at UCLA. Present address: Department of Human Anatomy, College of Medicine, Texas A&M University, O h E. Teague Research Center, College Station, Texas 77843.

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cal study and 31 in t h e neuroanatomical study. The details of t h e electrophysiological study were given in a previous publication (Rieke and Wenzel, '75). Briefly, t h e bird was anesthetized with Equithesin (in 500 ml, chloral hydrate 21.5 g, pentobarbital 4.86 g, magnesium s u l f a t e 10.63 g, propylene glycol 42.8%, ethyl alcohol 11.5%)( 2 ml/kg, imJ and placed in a stereotaxic frame. The skull over t h e dorsal telencephalon and olfactory bulbs was carefully removed. Stainless steel bipolar stimulating electrodes (resistances 100-150 K12) were placed into t h e olfactory bulb. The bulb was stimulated with electrical pulses of controlled duration, frequency and voltage strength. The forebrain was systemically explored with a stainless steel recording electrode (resistance 150-200 K Q ) . The evoked responses were amplified, displayed on a n oscilloscope and photographed. The location of t h e recording electrode in a n active site was marked by t h e method of Green ('58J, and confirmed histologically. The neuroanatomical technique used was a modified Fink-Heimer (I) procedure for t h e detection of degenerating axon cylinders and terminals (Fink and Heimer, '67). The animals were anesthetized with Equithesin (2 ml/kg, im) and placed in a stereotaxic frame. MATERIALS AND METHODS The olfactory bulbs in t h e pigeon are situated Two different experimental techniques, (1) on either side of t h e mid-sagittal plane a t the electrophysiological and (2) neuroanatomical, caudal and superior aspect of t h e orbits. They were used to determine those areas of t h e a r e covered by a large sinus and the bony pigeon brain t h a t receive projections from t h e calvarium. A lesion was placed in one olfacolfactory bulbs. Fifty-seven adult pigeons of tory bulb in t h e following manner. The outer either sex were used in t h e electrophysiologi- table and medullary bone were removed over

tral organization of this system. In its external gross features i t resembles that of other vertebrates. Neuroepithelial receptor cells are located in t h e olfactory mucosa. Axons of these neurons form t h e olfactory nerves t h a t terminate in t h e bulbs (Bang, '60, '71; Ariens Kappers et al., '60; Nieuwenhuys, '67; Andres, '70; Graziadei and Metcalf, '71; Oley e t al., '75). The olfactory bulbs of birds vary in size from order to order (Bang and Cobb, '68; Bang, '71) and are laminated even in those species with small fused bulbs, like t h e sparrow (Huber and Crosby, '29). The connections of t h e pigeon olfactory bulb with areas of t h e forebrain were examined for several reasons. The pigeon has been used in many of the behavioral and electrophysiological studies t h a t suggest olfaction is a functional sensory system. Though t h e pigeon brain is probably t h e most extensively studied of all avian brains, i t was not until recently (Rieke and Wenzel, '75) t h a t any avian olfactory system had even been examined experimentally. In this paper we will describe t h e forebrain projections of t h e pigeon olfactory bulb, including a brief review of t h e ipsilateral projections reported by Rieke and Wenzel ('75).

Abbreviations Ac, nucleus accumbens

A, archistriatum Ba, nucleus basilis BO, bulbus olfactorius ca, commissura anterior CPP, cortex prepiriformis CV, cerebral vein E, ectostriatum EPL, external plexiform layer fa, tractus fronto-archistriatalis fpl, fasciculus prosencephali lateralis GCL, granule cell layer GI,, glomerular layer HA, hyperstriatum accessorium HD, hyperstriatum dorsale HP, hippocampus HV, hyperstriatum ventrale INP, nucleus intrapeduncularis IPL, internal plexiform layer Ifm, lamina frontalis suprema

lfs, lamina frontalis superior Ih, lamina hyperstriatica Imd, lamina medullaris dorsalis LPO, lobus parolfactorius ML, mitral cell layer MSS, mid-sagittal sinus N, neostriatum NC, neostriatum caudale om, tractus occipitomesencephalicus ON, olfactory nerve ON BV, olfactory nerve blood vessel OS, olfactory sinus PA, paleostriatum augmentatum PC, paleostriatal complex PP, paleostriatum primitivum PVL, periventricular layer qf, tractus quintofrontalis S, nucleus septalis tsm, tractus septomesencephalicus v, ventriculus

PIGEON OLFACTORY SYSTEM

Fig. 1 The relations between the olfactory nerves, rostra1 forebrain and t h e venous drainage are shown in part A. The forebrain and the olfactory nerves (ON) are stippled. The olfactory sinus (0s)and its feeder vessels are colored black. Rostra1 is up in part A. Part B is a photomicrograph of a coronal section of the laminated olfactory bulb, the cortex prepiriformis and a small portion of the hyperstriatum ventrale. Part C is a coronal section through the forebrain a t A: 8.25 mm (atlas of Karten and Hcdos, '67) and demonstrates the relations between the lobus parolfactorius (LPO) and a portion of the paleostriatal complex.

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t h e sinus above t h e olfactory bulb. The thin inner table was carefully picked off of t h e sinus under a dissecting microscope. A flat metal lance with one blunt and one sharp edge was constructed from a scalpel blade. The lance was heated in a Bunsen burner and then passed through t h e middle portion of t h e exposed sinus with t h e blunt edge oriented towards t h e midsagittal plane. A portion of the olfactory bulb was scooped out on t h e flat face of t h e lance. Typically t h e rostral and lateral portions of t h e olfactory bulb were ablated. The parts of t h e bulb remaining after t h e lesion consisted of a thin medial strip, t h e postero-medial portion and t h e small olfactory peduncle (fig. 3). Bleeding from t h e sinus was minimized by use of the heated lance and it stopped rapidly when powdered Gelfoam was placed in t h e exposed area. The wound was sutured closed. Four sham-operated control animals were used to determine how much of t h e observed degeneration in the experimental animals was due to disruption of the venous sinus over the bulb. The controls were treated in the same manner as t h e experimental animals except that only t h e sinus was opened. The experimental birds were sacrificed according to a schedule of post-operative survival periods ranging from six hours to ten days. Fibers undergoing degeneration follow a specific time course. There is a beginning, a period of maximal degenerative changes and a period where all signs of degenerating fibers disappear (Ebbesson, ' 7 0 ) . Fibers degenerate rapidly in t h e pigeon and t h e best patterns of degeneration were seen 24 hours following t h e lesion to t h e olfactory bulb. Two of t h e shamoperated controls were sacrificed one day after t h e sinus was opened, one animal was sacrificed at two days, and another at six days. Each bird was deeply anesthetized, exsanguinated and perfused with 10%)formalin. Any animal showing evidence of subdural or epidural hemorrhage was discarded. The brain was stored in t h e fixative for two weeks, was then completely removed from t h e skull, and placed in a mixture of 35Y) sucrose and 10% formalin for a t least one week. A cut in t h e coronal plane was made through t h e cerebellum providing a stable base upon which t h e brain could rest. The brain was divided in t h e midsagittal plane to give one intact half and one with t h e lesion. Each half was placed on t h e freezing head of a cryostat and sections of 25 Fm thickness were cut in t h e coronal or

sagittal plane. Sections were collected in a manner t h a t produced five serial sets, each set consisting of every fifth section (e.g., 1, 6, 11, etc. or 2, 7, 12, etc.). One set for each survival period was stained with carbolfuchsin, t h e remaining sets were processed by a modified Fink-Heimer I technique. The sections were examined under oil and t h e degeneration patterns were plotted on drawings of enlargements of t h e actual sections. Twenty-six brains from unoperated pigeons were examined to provide details of t h e normal anatomy of t h e pigeon olfactory system. These birds were anesthetized and perfused with 10%formalin. The brains were embedded in paraffin and cut a t 10 p m in either t h e coronal or sagittal plane. The serial sections were mounted and stained with carbolfuchsin and luxol-fast blue. RESULTS

Normal anatomy The olfactory receptors are situated in t h e olfactory mucosa and t h e axons of these neuroepithelial cells aggregate to form two olfactory nerves. These nerves approach each other a t a point in t h e bony region between the superior and rostral parts of t h e orbits (fig. 1A). Here they enter a n incomplete bony canal which passes superiorly between the orbits. The two nerves remain in close proximity up to t h e site where they end in the olfactory bulb. The paired olfactory bulbs are covered superiorly by a venous sinus (0s).This sinus receives blood from two large cerebral veins (CV) t h a t course along t h e lateral surface of t h e hemisphere between t h e hyperstriatal mass and neostriatum. A midsagittal sinus (MSS) also empties into t h e sinus over t h e olfactory bulbs. Two small vessels coursing along t h e olfactory nerves terminate in t h e sinus (ON BV). The sinus over the bulbs is opened in t h e process of placing a lesion in one olfactory bulb. It is therefore important to examine those areas of t h e brain t h a t may contribute blood to t h e sinus for evidence of degeneration attributable to damage of their vascular supply rather than to t h e olfactory bulb. Figures 1B and 1C illustrate t h e parts of t h e forebrain associated with t h e olfactory system. The nomenclature used is t h a t found in t h e atlas of t h e pigeon brain by Karten and Hodos ('67). The olfactory bulb (fig. 1B) has seven layers: (1)olfactory nerve (ON), (2) glo-

PIGEON OLFACTORY SYSTEM

45

6 ,

\

12-

L2.5\

\\

Fig. 2 Type I responses are demonstrated on t h e right side of the figure. These responses were obtained from (a) the hyperstriatum ventrale (HV), (b) the cortex prepiriformis (CPP) and (c) the lobus parolfactorius (LPO), following stimulation of the ipsilateral olfactory bulb (BO, d). Calibration bars: voltage bar for a. 50 p V , and for b and c, 100 FV; time bar for a, b and c, 20 msec.

merular layer (GL) with periglomerular or external granule cells, (3) external plexiform layer (EPL), (4)mitral cell layer (ML), (5) internal plexiform layer (IPL), (6)granule cell layer (GCL), (7) t h e ependymal or periventricular layer (PVL). The olfactory ventricle is relatively large. The mitral cells a r e of moderate size (10-15 pm diameter cell body) and form a recognizable lamina. A few large neurons are also located in the external plexiform layer. The principal dendrite of t h e mitral cell is directed peripherally into t h e glomerular layer where synaptic connections are made upon i t by t h e axons of t h e olfactory receptor neurons (Andres, '70). The axon of t h e mitral cell is small, ranging in diameter from 0.5 p m to 1.9 pm. These small axons do not aggregate to form discrete olfactory striae (Rieke and Wenzel, '75) in contrast with mammals and reptiles (Heimer, '69). Axons of mitral cells course through t h e forebrain in a loosely organized fashion. They do reach t h e small olfactory cortex. The prepiriform cortex based on electrophysiological and experimen-

tal neuroanatomic observations, is t h e olfactory cortex (Rieke and Wenzel, '75; Macadar et al., '75). This cortex comprises less than 2%) of t h e surface of one hemisphere. The avian forebrain has not developed a n elaborate cortex; rather, i t is organized around neuronal masses (Nauta and Karten, '70). The neural masses in which t h e small axons of mitral cells terminate a r e illustrated in the line drawing of figure 1C. The lamina hyperstriatica (lh) separates t h e hyperstriatum ventrale (HV) dorsal to t h e lamina from t h e neostriatum (N) ventral to the lamina. The boundary between t h e neostriatum and t h e underlying paleostriatal complex is t h e lamin a medullaris dorsalis (lmd). The ectostriat u m (El is situated upon t h e lmd lateral to t h e neostriatum. Inferior to t h e lmd are located two large nuclear masses: laterally t h e paleostriatal complex and medially t h e lobus parolfactorius. The paleostriatal complex (PC) is composed of t h e paleostriatum augmentatum (PA), t h e paleostriatum primitivum (PP) and t h e nucleus intrapeduncularis

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NB25 1D

NB28 4 0

Fig. 3 A post-mortem evaluation of the magnitude of the lesion to the left olfactory bulb in birds NB 25 and NB 28 is provided here. Bird NB 25 was sacrificed one day and NB 28 four days after the lesion. The remains of the left olfactory bulb included a thin medial strip, the posteromedial portion and the small peduncle. The right bulbs are intact.

(INP). Medially, t h e paleostriatum augmentatum merges with t h e lobus parolfactorius (LPO) (Karten and Dubbeldam, '73). EXPERIMENTAL RESULTS

Efferent fibers from one olfactory bulb terminate in both sides of t h e forebrain. Therefore the olfactory projection field will be described under two subdivisions: (1)uncrossed and (2) crossed. The uncrossed component has been reported in a previous paper (Rieke and Wenzel, '75) and only t h e salient features will be presented here.

Uncrossed field Electrophysiological observations Electrical stimulation of t h e olfactory bulb evoked two classes of responses from the ipsilateral forebrain (Rieke and Wenzel, '75). They were designated as Type I or Type I1 responses. Type I1 responses were recorded from areas connected to t h e olfactory bulb by a polysynaptic pathway, viz., a t least two neurons between t h e bulb and t h e area of t h e active response. Type I responses were recorded from those areas t h a t receive direct olfactory inputs specifically, t h e hyperstriatum ventrale (HV), lobus parolfactorius (LPO) and

t h e cortex prepiriformis (CPP) (fig. 2). The type I responses consisted of a prominent positive wave t h a t develops rapidly and is followed by a low amplitude negative wave of long duration. The onset latency ranged from 4-8 msec. The responses could be driven repetitively up to 20-25 Hz, which was the highest rate of effective stimulation for all olfactory evoked responses. A post-tetanic depression period of up to 30 seconds was also observed. The short onset latency, the high r a t e of effective repetitive stimulation and the presence of post- tetanic depression are physiological indicators of a monosynaptically evoked response (Rieke and Wenzel, '75). The HV, LPO and CPP have been confirmed as t h e primary olfactory centers of t h e pigeon in additional electrophysiological studies (Macadar et al., '75; Hutchison et al., '77). Neuroanatomical observations Degenerating fibers and axon terminals were found in t h e cortex prepiriformis (CPP), lobus parolfactorius (LPO) and the hyperstria t u m ventrale (HV) following lesions in t h e ipsilateral olfactory bulb (Rieke and Wenzel, '75). The post-mortem controls of t h e lesions in t h e bulb for each animal to be described are demonstrated in figure 3. The olfactory nerve was cut as a result of t h e lesion to the olfactory bulb. Fine fragments of t h e degenerating olfactory nerve were located over the glomeruli remaining in t h e olfactory bulb (fig. 4a). The fragments a r e very fine because the normal axons comprising the olfactory nerve have a diameter of 0.1 p m to 0.5 pm. The patterns of degeneration in the forebrain were well established by 24 hours after t h e lesion to t h e ipsilateral olfactory bulb. Three loosely organized groups of fragmented fibers were observed (fig. 5). Two of these groups, one to t h e cortex prepiriformia (CPP) and t h e second to t h e hyperstriatum ventrale (HV) were uncrossed. Fragmented fibers and terminals were scattered through the LPO. Fibers were traced t h r o u g h t h e nucleus accumbens (Ac) into t h e anterior commissure and to t h e opposite forebrain. The HV, CPP and LPO contained degenerating axon cylinders and terminals four and eight days after lesions to t h e bulb (fig. 6).No additional areas outside of t h e HV, CPP, Ac, LPO, were found to contain degenerating axons or terminals. Qualitatively t h e intensity of degeneration was t h e same, one to four days after t h e lesion,

PIGEON OLFACTORY SYSTEM

Fig. 4 Photomicrographs of sections through the olfactory nerve, the glomerular (GL) and external plexiform layers (EPL) from an experimental animal (A, B) and a sham-operated control (C). The experimental animal was sacrificed 24 hours after a lesion was made in the left olfactory bulb. Note the many silver grains over the shrunken glomeruli on the lesion side in A in contrast to the few grains in B (normal side) and in t h e sham-control (0.The pial surface is up. Calibration lines 50 pm.

NB-25

LS

NS

Fig. 5 A line drawing of coronal sections from NB 25 t h a t represents the pattern of degeneration 24 hours after a lesion to t h e left olfactory bulb. The first seven drawings starting in t h e upper left hand corner are from the left side of t h e brain. The remaining seven drawings are from the right side (NS) of the brain. The coordinates provided with each section (e.g., 14.5, 14.10, etc.) indicate the approximate levels from the atlas of Karten and Hodos (‘67). The sections are not evenly spaced.

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NB-28

Fig. 6 A line drawing of sagittal sections from NB 28 t h a t represents the pattern of degeneration four days after a lesion to the left olfactory bulb. The upper row shows the degeneration on the side of the brain with the lesion (LS) while the lower row of drawings shows the degeneration seen on the right side of the The coordinates (1.5, 2.5, etc.) indicate the approximate lateral plane of section in reference t o brain (NS). the atlas of Karten and Hodos ('67).

and it appeared to decline after the fourth day. Brains of the sham-operated control were free of degenerating axon cylinders or terminals that could be attributed to vascular damage. A few silver grains were found over the glomeruli in the bulb under the opened olfactory sinus (fig. 4c). Those areas which are directly in contact with the olfactory sinus and its tributaries, in particular the hyperstriatum accessorium (HA), hyperstriatum ventrale (HV) and the neostriatum (N), contained no evidence of degenerating fibers or terminals in the sham-operated control animals.

Crossed field Degenerating axons and terminals were found in the opposite forebrain as early as 18 hours after the lesion to the olfactory bulb. The degeneration patterns were well established after 24 hours (fig. 5). There were only a few silver grains over the glomeruli of the contralateral olfactory bulb a t 24 hours in the experimental and sham-control animals (figs. 4b,c). Degenerating axon fragments and terminals were observed predominantly in the paleostriatal complex, particularly the paleo-

striatum primitivum (PP), and in the caudal portion of the lobus parolfactorius. Fragmented fibers spread from the anterior commissure (ca) into the contralateral nucleus accumbens (Ac) on their course to t h e paleostriatal complex and the lobus parolfactorius. A few fragments and possible terminals were seen in the paleostriatum augmentatum (PA) and the more rostra1 portions of the LPO. Degenerating axon terminals (t and arrows in fig. 7) and argyrophilic granules (probably collateral terminals) were found on the cell bodies and processes of many of the large neurons of the contralateral PP (fig. 7 ) . The paleostriatum primitivum consists of large multipolar neurons distributed in a dense feltwork (neuropil) of nerve cell processes. The degenerating fibers in the neuropil had fusiform swellings and the irregular beaded appearance described by Ebbesson ('70) of degenerating axons and synaptic endings in non-mammalian species (fig. 7B, neurons C,, C1,CJ. The paleostriatum primitivum on the side of the brain with the olfactory bulb lesion was devoid of degenerating terminals and argyrophilic granules. Some of the fibers

PIGEON OLFACTORY SYSTEM

49

Fig. 7 A three-part composite of photomicrographs of t h e right paleostriatum primitivum from a bird in which t h e left olfactory bulb had sustained a lesion 24 hours prior to sacrifice. A, low magnification view of a portion of t h e paleostriatum primitivum; B, a photomontage of t h e large neurons around the blood vessel; C, degenerating terminals (t and arrows) seen on t h e cell bodies or processes of neurons only on t h e side opposite t h e lesion; neurons C,, C, and C,, high power photomicrographs of neurons 1, 2 and 3 in part A.

in the neuropil had fusiform swellings (fig. 8). The sham-operated control showed no degenerating axons or terminals in either (PP) (fig. 9) ; in fact the control showed no significant degeneration in any portion of the forebrain.

No evidence of degenerating terminals or fibers of passage was seen in the stria medullaris, habenular nuclei or the small habenular commissure. The hypothalamus, archistriatum, neostriatum and the cortex prepiriformis

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GARL KALMAN RIEKE A N D BERNICE M. WENZEL

Fig. 8 A three-part composite of photomicrographs of the left paleostriatum primitivum from a bird in which the left olfactory bulb had sustained a lesion 24 hours prior to sacrifice. A is a low-magnification view of a portion of the paleostriatum primitivum. B is a photomontage of the large neurons around the blood vessel. The arrow marks a rare neuron with argyrophilic granules. In C no degenerating terminals are seen. The neurons appear dark, but are not pyknotic since discrete nuclei and nucleoli are present. Neurons numbered 1, 2 and 3 are the same in parts A, B and C.

PIGEON OLFACTORY SYSTEM

Fig. 9 A three-part composite of photomicrographs of the right paleostriatum primitivum of a sham-operated control in which the sinus over the left olfactory bulb was punctured 24 hours prior to sacrifice. A is a low-magnification view of a portion of the paleostriatum primitivum. B is a photomontage of the large neurons around the centrally placed blood vessel. The region of neuropil without nerve cells between the two parallel broken lines in A was deleted in B to save space, The large arrows in B mark the line of apposition of these two surfaces after the deletion. C shows three representative neurons demonstrating the lack of degenerating terminals and argyrophilic granules. The neurons numbered 1, 2 and 3 are the same in parts A, B and C.

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(the olfactory cortex) were devoid of degenerating terminals or axons of passage. The patterns of degeneration after four and eight days were similar to the pattern after one day. The intensity of degeneration was qualitatively the same for one and four days, and reduced by eight days. Degeneration was restricted to areas ventral to the lamina medullaris dorsalis (lmd) (fig. 6). Fragmented axons were traced from the anterior comxissure into predominantly the caudal LPO and the paleostriatum primitivum. A small number of neurons in the paleostriatum augmentatus (PA) had degenerating terminals on their cell bodies and processes. Large neurons in the P P were found with degenerating terminals on their cell body or processes. The P P and PA on the side of the brain with the lesion to the olfactory bulb were free of degenerating terminals. The sham-operated controls showed no degenerating terminals in either the right or left paleostriatal complex. The centers of the dorsal ventricular ridge (N, E, HV) were free of degeneration in the experimental animal. No degenerating terminals or fragmented axons were found in the CPP, HA, A or hypothalamus. DISCUSSION

Difficulties inherent to definition of pigeon olfactory system Three items posed potential problems in the analysis of our experimental observations. These included (1) the small diameter of the secondary olfactory fibers, (2) the presence of a large vascular sinus over the bulb and (3) possibility of damage to the anterior olfactory nucleus and the accessory olfactory bulb. Interpretation of the experimental data without considering these items, could give a distorted view of the pigeon olfactory system, particularly since there was no experimentally determined model of any avian olfactory system available for comparison. The axons of mitral cells are small (0.5 pm1.9 pm) and form the principal pathways leaving the pigeon olfactory bulb. The possibility existed that because of their small diameter, degenerating mitral cell axons and a portion of their terminal fields might not be visible with the light microscope (Ebbesson, '70). Two different experimental methods, (1) electrophysiological and (2) neuroanatomical, were used to resolve this problem. Our electrophysiological and neuroanatomical observations

were complementary and led us to conclude that the cortex prepiriformis (CPP), hyperstriatum ventrale (HV) and lobus parolfactorius (LPO) comprised the ipsilateral olfactory projection field (Rieke and Wenzel, '75). Hutchison et al. ('771, using the methods of averaged evoked potentials (AEP) and poststimulus time histograms (PST), confirmed our observations that the CPP, HV and LPO receive secondary olfactory fibers. The olfactory bulb is covered by a vascular sinus and the question arises as to how much of the degeneration observed is due to vascular interference following damage to the sinus. The few silver grains found in the glomeruli of the sham-operated controls and the contralateral olfactory bulb of the experimental animals might have been due to vascular trauma. More probably they resulted from the continuous renewal or reconstitution of the olfactory receptor neurons (Graziadei and Metcalf, '71; Oley et al., '75). The lack of degenerating axons and terminals in the forebrain of the sham-operated controls strongly suggests that damage to the sinus did not contribute significantly to our results. The degeneration patterns produced by lesions to the olfactory bulb were devoid of any contributions from an accessory olfactory bulb or the anterior olfactory nucleus. Avian species do not have an accessory olfactory bulb (Ariens Kappers et al., '60; Nieuwenhuys, '67). Furthermore, while little accord exists as to the precise locus and size of the anterior olfactory nucleus in the bird (Crosby and Humphrey, '39; Zeier and Karten, '73), the stereotaxic atlas of Karten and Hodos ('67) places this small nucleus a t the postero-latera1 extreme of the bulb (AP: 13.75 mm). None of our lesions to the bulb extended into the region of the presumptive anterior olfactory nucleus. Sources o f secondary olfactory fibers The mitral cell is one neuron found in the olfactory bulbs of many different vertebrates (Ariens Kappers et al., '60; Nieuwenhuys, '67). Its principal dendrite projects into the glomerular zone and on this dendrite, terminate the axons of the olfactory nerve (Andres, '70). The mitral cell is the principal second neuron of the olfactory system and its axon connects the bulb with other parts of the forebrain (Ariens Kappers et al., '60; Nieuwenhuys, '67; Andres, '70; Shepherd, '72). In the pigeon, it is

PIGEON OLFACTORY SYSTEM

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other vertebrates in contrast to the pigeon, secondary olfactory fibers may cross in the habenular commissure (Scalia et al., '69; Heimer, '69; Royce and Northcutt, '691, both t h e anterior and habenular commissures (Finger, '75) or they may not enter the contralateral hemisphere at all (Ebbesson and Heimer, '70; Braford and Northcutt, '73). As our results indicate, the LPO is the only region to receive fibers from both olfactory bulbs. Zeier and Karten ('71) and Karten and Dubbeldam ('73) have demonstrated that the pigeon LPO projects to t h e ipsilateral lateral hypothalamus through the fasciculus prosencephali medialis (medial forebrain bundle of pigeon). The hypothalamic projection seems significant in view of the involvement of t h e hypothalamus in the regulation of visceral responses. These connections might account for the observed changes in heart and respiratory rates of pigeons upon presentation of odors (Wenzel and Salzman, '68; Wenzel and Sieck, '72). The paleostriatal complex is the other area t h a t receives secondary olfactory fibers through the anterior commissure. As our observations indicate, this complex has two sources of olfactory afferents: (1) a direct Olfactory projection fields and input from the contralateral bulb to the P P their interconnections and (2) an indirect input from the ipsilateral The cortex prepiriformis (CPP) and the bulb to the PA with a relay in the ipsilateral hyperstriatum ventral (HV) have as part of hyperstriatum ventrale. These two sources their afferent connections secondary olfactory may have opposite effects upon neurons of the fibers from the ipsilateral bulb (Rieke and paleostriatal complex. Stimulation of the ipsiWenzel, '75). The connections of the small lateral bulb was seen to inhibit unit activity olfactory cortex (CPP) with other areas of the in the P P (Rieke, unpublished observation; brain have not yet been defined, nor are those Hutchison et al., '771, while stimulation of the of the HV completely resolved. The HV pro- contralateral olfactory nerve increased the jects onto neurons of the ipsilateral paleo- frequency of discharge of neurons in the striatum augmentatum (PA) (Zeier and Kar- (PP) (Hutchison, personal communication). ten, '731, a portion of the paleostriatal complex Karten and Dubbeldam ('73) have proposed (Karten and Dubbeldam, '73). Nerve cells of the that the paleostriatal complex of the pigeon paleostriatal complex can therefore be affected may be the avian homology of the mammalian by signals from the ipsilateral olfactory bulb basal ganglia, part of a n ancient motor system. The function of the paleostriatal complex following a relay in the HV. Our finding that the anterior commissure in birds is not known. However, if i t is carries secondary olfactory fibers to the lobus involved in motor activities, this might shed parolfactorius (LPO) and the paleostriatum some light on the role played by the olfactory primitivum (PP) is given further support in a system in homing or navigation (Papi et al., recent study on the connections of the anteri- '71, '72; Grubb, '74; Baldaccini et al., '75). or commissure by Zeier and Karten ('73). ACKNOWLEDGMENTS According to these authors the LPO and The authors wish to thank Doctor Noel de paleostriatal complex are two areas t h a t form a portion of the projection field of the pars bul- Terra, and Doctors A. B. and M. E. Scheibel for baris of this commissure in the pigeon. In their helpful criticisms and suggestions in the

possible that fibers leaving the bulbs are not exclusively mitral cell axons because processes of various cell types (stellate, transitional, large and small goblet and tufted) have been described as leaving the olfactory bulbs of other vertebrates (Nieuwenhuys, '67). Golgi studies would be appropriate to determine the types of neurons in the bulb, especially those in the granule cell layer. Unfortunately such studies are lacking in the pigeon and it is not known whether neurons in the granule cell layer send processes to areas outside of the bulb. As in other vertebrates, mitral cells form a prominent lamina in t h e pigeon bulb (Andres, '70). There are also a few neurons in the external plexiform layer which might be interpreted as displaced mitral cells or tufted cells (Crosby and Humphrey, '39; Nieuwenhuys, '67). Tufted cells, and in particular, the projections of their axons, are still perplexing even in mammals (Cajal, '11; Valverde, '65; Shepherd, '72). The neurons whose processes form the efferent paths from the pigeon bulb need to be identified in future studies using the method of retrograde intraaxonal transport of horseradish peroxidase (LaVail et al., '73).

54

GARL. KALMAN RIEKE AND BERNICE M. WENZEL

preparation of this manuscript. We wish to thank Ms. C. Stroman for her typing of t h e manuscript. We are grateful for t h e technical skills of Irene Jones in preparation of many of t h e histological sections and t o Harold McCaffery, Jim Michaud and Pan Cummings for their assistance. LITERATURE CITED Andres, K. H. 1970 Anatomy and ultrastructure of the olfactory bulb in fish, amphibia, reptiles, birds and mammals. In: Taste and Smell in Vertebrates. G. E. W. Wolstenholme and J. Knieht. eds. J. A. Churchill. London, pp. 177-196. Ariens Kappers, C. U , G. C. Huber and E. C. Crosby 1960 The Comparative Anatomy of the Nervous System~ofVertebrates, Including Man. Vol. 111. Hafner Publishing Co., New York, pp. 1358-1401. Baldaccini, N., S. Benvenuti, V. Fiaschi and F. Papi 1975 New data on the influence of olfactory deprivations on the homing behavior of pigeons. In: Olfaction and Taste V . D. Denton and J. P. Coghlan, eds., Academic Press, New York, pp. 351-353. Bang, B. G. 1960 Anatomical evidence for olfactory function in some species of birds. Nature, 288: 547-549. 1971 Functional anatomy of the olfactory system in 23 orders of birds. Acta Anat. (Suppl. 58),79: 1-76. Bang, B. G., and S. Cobb 1968 The size of the olfactory bulb in 108 species of birds. Auk, 85: 55-61. Braford, M. R., and R. G. Northcutt 1973 Olfactory bulb projections in the bichir,PoZypterus. J. Comp. Neur., 156: 165-178. Cajal, S. Ramon y 1911 Histologie du Systeme Nerveux de I'Homme et des Vertebres. Vol. 11. Maloine, Paris. Crosby, E. C., and T. Humphrey 1939 Studies of the vertebrate telencephalon. I. The nuclear configuration of the olfactory and accessory olfactory formations and the nucleus olfactorius anterior of certain reptiles, birds and mammals. J. Comp. Neur., 71: 121-213. Ebbesson, S. 0. E. 1970 The selective silver-impregnation of degenerating axons and their synaptic endings in nonmammalian species. In: Contemporary Research Methods in Neuroanatomy. W. J. H. Nauta and S. 0. E. Ebbesson, eds. Springer-Verlag, New York, pp. 132-161. Ebbesson, S. 0. E., and L. Heimer 1970 Projections of the olfactory tract fibers in the nurse shark (Ginglymostoma cirratuml. Brain Res., 27: 47-55. Finger, T. E. 1975 Distribution of olfactory tracts in bullhead catfish fZctalurus nebulosusi. J. Comp. Neur., 162: 125-141. Fink, R. P., and L. Heimer 1967 Two methods for selective silver impregnation of degenerating axons and their synaptic endings in the central nervous system. Brain Res., 4: 369-374. Graziadei, P. P. C., and J. F. Metcalf 1971 Neuronal dynamics in the olfactory mucosa of the adult vertebrates. Anat. Rec., 269: 328. Green, J. D. 1958 A simple microelectrode for recording from the central nervous system. Nature, 182: 962. Grubb, T. C. 1974 Olfactory navigation to the nesting burrow in Leach's petrel (Oceanodroma leucorhoa). Anim. Behav., 22: 192-202. Heimer, L. 1969 The secondary olfactory connections in mammals, reptiles and sharks. Ann. N. Y. Acad. Sci., 167: 129-146. Henton, W. W., J. C. Smith and D. Tucker 1966 Odor discrimination in pigeons. Science, 153: 1138-1139. Y

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PIGEON OLFA(OTORY SYSTEM Stager, K. W. 1964 The role of olfaction in food location by t h e turkey vulture (Cathartes aura). L. A. County Museum Contributions in Science. No. 81, pp. 1-63. Tucker, D. 1965 Electrophysiological evidence for olfactory function in birds. Nature, 207: 34-36. Valverde, F. 1965 Studies on the Piriform Lobe. Harvard University Press, Cambridge. Wenzel, B. M. 1968 The olfactory prowess of the kiwi. Nature, 220: 1133-1134. 1971 Olfactory sensation in the kiwi and other birds. Ann. N. Y. Acad. Sci., 188: 183-193.

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Wenzel, B. M., and A. Salzman 1968 Olfactory bulb ablation or nerve section and pigeons’ behavior in non-olfactory learning. Exp. Neurol., 22: 472-479. Wenzel, B. M., and M. H. Sieck 1972 Olfactory perception and bulbar electrical activity in several avian species. Physiol. Behav., 9: 287-293. Zeier, H. J . , and H. J. Karten 1971 The archistriatum of the pigeon: organization of afferent and efferent connections. Brain Res., 31: 313-326. 1973 Connections of the anterior commissure in the pigeon fColurnba h i d . J. Comp. Neur., 150: 201-210.