electrophysiological evidence for a topographical

Electrophysiological Evidence for a Topographical Projection of the Nasal Mucosa onto the Olfactory Bulb of the Frog

R I C H A R D M. C O S T A N Z O and M A X W E L L M. MOZELL

From the Department of Physiology, State University of New York, Upstate Medical Center, Syracuse, New York 13210. Dr. Costanzo's present address is The Rockefeller University, New York 10021.

A B S T R A C T Three olfactory nerve branches respectively subserving either a medial, an intermediate, or a lateral region of the dorsal olfactory receptor sheet of the bullfrog Rana catesbeiana were electrically stimulated with bipolar platinum hook electrodes. Extracellular single unit responses from 93 second-order cells in different regions of the olfactory bulb were recorded with metal-filled glass micropipets. The excitatory responsiveness of each unit to the stimulation of each of the three nerve branches (response profile) was determined. Some units were sensitive to stimulation of each of the three nerve branches, thus suggesting a wide projection from the entire receptor sheet. On the other hand, other units were more selective. Of this latter group, units in the lateral bulb were excited by nerve branches subserving the more lateral regions of the receptor sheet; units in the medial bulb were excited by the nerve branches subserving the more medial regions of the receptor sheet. These data provide electrophysiological evidence for a topographical projection of the olfactory receptor sheet onto the olfactory bulb, and further suggest that the projections onto different bulbar cells vary in degree of localization.

I N T R O D U C T I O N

On the basis of his recordings o f mult iunit discharges in d i f ferent anterior-posterior regions o f the olfactory bulb in rabbit and cat in response to different odorants , Adr ian (1951, 1953, and 1954) in t roduced the concept o f differential spatial and tempora l patterns o f neural activity as one o f the mechanisms that could underl ie odoran t discrimination. Adrian reasoned that the di f ferent spatial and temporal patterns he observed at the level of the olfactory bulb must reflect similar precursory spat iotemporal patterns o f neural activity at the level o f the olfactory receptor sheet. A subsequent histological investigation using re t rograde degenera t ion techniques (Le Gros Clark, 1951) reversed the conclu- sion o f an earlier, less extensive study (Le Gros Clark and Warwick, 1946) by demons t ra t ing at least a loose topographical projection o f the olfactory epithelium onto the olfactory bulb. Al though this projection was found to be more precise along a dorsoventral axis than in the anteroposter ior direction empha-

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298 THE JOURNAL OF GENERAL PHYSIOLOGY'VOLUME 6 8 ' 1976

sized by Adrian and although some of the projections seemed rather diffuse, this histological evidence did, nevertheless, give credence to the concept of a topographical representation of the epithelium in the bulb. More recent studies using modern anterograde tracing techniques (Land et al., 1973; Land and Shepherd, 1974) have given fur ther support for the existence of a topographical projection, albeit along spatial directions not emphasized by Adrian. In these recent studies the dorsal recess of the nasal cavity was found to project to the dorsal aspect of the bulb whereas the lateral region of the nasal cavity projected to the lateral part of the bulb. In addition, these studies showed that there were regional differences in the degree of "sharpness" of these projections, ranging from precise to rather diffuse.

Freeman (1974) chose to emphasize the diffuseness of the projection of the mucosa onto the bulb while still acknowledging some topographic organization. This emphasis was based upon his analysis of evoked bulbar potentials produced by the electrical stimulation of a pool of olfactory nerve axons. Since each of the evoked potentials could be recorded over much of the bulbar surface, he concluded that the receptors in each part of the mucosa must influence glomer- uli over very broad regions of the bulb. Nevertheless, Freeman did observe that changes in the position of the stimulating electrodes in the population of olfactory nerve axons did produce shifts in the epicenters of these widely recorded evoked potentials. He therefore concluded that on the average there is a topographic organization of the population of olfactory nerve axons but not of the point-to-point variety.

From Adrian's work two mechanisms by which different spatiotemporal patterns might be established at the level of the receptor sheet are suggested: (a) the receptor cells might have differential selective sensitivities to different odorants (Gesteland et al., 1963; Mathews and Tucker, 1966; Mathews, 1972; O'Connell and Mozell, 1969) and receptors with like sensitivity might be clustered together into regional aggregates across the receptor sheet (Kauer and Moulton, 1974); (b) regardless of the selective sensitivity of the receptors per se, different patterns of neural activity might result as a consequence of differences in the sorption of odorant molecules across the mucosal sheet. Those odorants which are differentially sorbed would give rise to different odorant distribution patterns across the olfactory mucosa and thus different patterns of activity across the receptor sheet. Either or both of these mechanisms could project different regional and temporal patterns of excitation into the olfactory bulb as different odorants pass over the receptor sheet.

Although Mozell and Pfaffmann (1954) and Mozell (1958) have confirmed the differential spatiotemporal patterns that Adrian observed in the bulb, there was for some time no direct evidence from the olfactory mucosa itself to support Adrian's suggestion of spatiotemporal patterns at the peripheral level. Such evidence has been presented by Mozell (1964, 1966, 1970). He compared, for different odorants, the responses of two branches of the olfactory nerve which sampled the activity of two widely separated regions of the olfactory mucosa. He observed that both the relative magnitude of the responses of these two nerve branches and the differences in their latencies varied characteristically from chemical to chemical. Mozell proposed that these various space-time activity

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COSTANZO AND MOZELL Projection of Nasal Mucosa onto Olfactory Bulb 299

pa t te rns ref lected the di f ferent ia l sorpt ion (and consequent ly the di f ferent ia l distribution) o f the molecules o f d i f fe ren t odoran t s across the mucosa . Mozell and his co-workers later p resen ted m o r e direct evidence for di f ferent ia l molecular distr ibutions by ch romatograph ica l ly measu r ing the mucosal re tent ion t imes of d i f fe ren t odoran t s in the intact olfactory sac (Mozell and Jagodowicz , 1973) and by m a p p i n g the dis tr ibut ion o f tr i t iated o d o r a n t molecules across the mucosa ( H o r n u n g et al., 1975).

In addi t ion, recent data have been in te rp re ted as evidence for regional aggregates o f r ecep to rs with similar selective sensitivities (Mustapar ta , 1970; Kauer and Moul ton, 1974). O f par t icular interest in this r ega rd is Kaue r and Moulton 's s tudy of single units in the olfactory bulb of s a l amander in response to puncta te s t imulat ion o f the olfactory mucosa. Some mucosal regions a p p e a r e d more responsive to some chemicals than did others . This result seemed consist- ent with the results o f Moul ton 's earl ier studies (1963, 1965, 1967) involving mult iuni t d ischarges in the rabbi t olfactory bulb. Using an a r ray o f e lect rodes record ing f r o m a mosaic o f posit ions in the bulb, Moul ton noted that the pa t t e rn o f discharge magn i tudes across this mosaic d i f fe red for d i f fe ren t chemicals. Moul ton later (P fa f fmann , 1969) i n t e rp re t ed these da ta as be ing based u p o n a mosaic o f epithelial regions in which the receptors o f any one region have the same sensitivity but the receptors in d i f fe ren t regions have d i f fe ren t sensitivities.

T h u s there a p p e a r s to be evidence for two mechan i sms which could under l ie the spa t io tempora l analysis o f odoran t s at the recep tor level, i.e. aggrega tes o f selectively sensitive receptors and different ia l molecular distr ibutions. I t is impor t an t to note, as indicated by Mozell (1971), that these two mechan isms are not mutual ly exclusive and could indeed opera te in concer t to p roduce a m u c h g rea te r r ange o f spa t io tempora l pa t te rns than could e i ther mechan i sm alone.

Regardless o f whe the r one or bo th o f the p roposed mechan i sms is responsible for the activity pa t te rns established at the mucosa, it seems critical to u n d e r s t a n d how the pa t te rn may be p rese rved and t r ans fe r r ed to cells at h igher levels o f the olfactory system. In o the r sensory systems such preserva t ion is accompl ished by the display o f a topographica l project ion of the recep to r sheet at more central levels o f the system (Mountcastle, 1957; Hube l and Wiesel, 1962, 1965, 1968). T h e p resen t expe r imen t s were des igned to test for the existence o f a similar topographica l project ion o f the olfactory recep to r sheet onto the second o r d e r neurons in the olfactory bulb.

M E T H O D S

Preparation

Louisiana bullfrogs Rana catesbeiana were immobilized with a subcutaneous injection of d- tubocurarine (30 mg/kg), wrapped in a moist towel, and placed in an ear-bar head holder to fix the head firmly in place. Anesthesia for all surgical procedures was provided by topical application of 5% procaine hydrochloride. The bone and cartilage overlying the olfactory bulbs and the dorsal aspect of the olfactory sac were removed. This allowed access to the branches of the olfactory nerve which splay out across the dorsal surface of the ~ olfactory sac (Fig. 1). Of these branches, three widely separated branches (viz., the most medial, an intermediate, and the most lateral) subserving different localized regions in the mucosa (Mozell, 1964) were dissected free from the surrounding connective tissue.

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These branches were lifted onto bipolar platinum stimulating electrodes (250/~m diam). The animal was grounded through a silver-silver chloride wire which contacted a piece of Ringer's-soaked cotton placed on the bone just posterior to the exposed olfactory bulbs. Electrical stimulation was obtained with a Grass $48 constant voltage stimulator equipped with a Grass stimulus isolation unit (SIU-5) (both from Grass Ins t rument Co., Quincy, Mass.). Metal-filled micropipets (Gesteland et al., 1959), having tip diameters of less than 5/~m, were used to record single uni t activity in the olfactory bulb. This microelectrode was capacitor coupled to a Grass P16 amplifier and the amplified signals were led to both a Tektronix 532 oscilloscope (Tektronix, Inc., Beaverton, Ore.) and a Grass AM-5 audio monitor. Photographic records were taken of the scope display with a Grass C-4 Kymo- graph camera.

Procedures Electrode penetrations were made within a band of coordinate positions which was found to optimize the possibility of encountering units in the mitral cell layer of the olfactory bulb (see section on Electrode Placement). Once a single bulbar unit was identified and its exact coordinate position noted, its responsiveness to the electrical stimulation of each of the three olfactory nerve branches was tested. For each bulbar unit each of the three nerve branches was stimulated with a series of test pulses 0.1 ms in duration and ranging in voltage from 10 to 60 V. (In preliminary experiments it was found that i fa unit did not respond at 60 V, it would not respond at higher voltages.) The inset of Fig. 1 represents a response typical of those recorded from these bulbar units after stimulation of an olfactory nerve branch. The response consists of a single driven spike superimposed on an evoked field potential. In order to emphasize the spike itself, signals were routinely filtered to selectively attenuate the field potentials. The analysis of the data to be presented is based upon the presence or absence of the driven spike only.

In Fig. 1 the interval between stimulus onset and the spike response was 110 ms. Since the distance between the stimulating and recording electrodes was approximately 1.5-2.0 cm, a conduction velocity of 0.14-0.18 m/s was estimated. This conduction velocity is comparable to values reported by Gasser (1956) for olfactory nerve fibers of the pike.

Control for the Spread of Stimulus Current

Since these experiments were designed to test the responsiveness of bulbar units to the stimulation of different nerve branches subserving different localized regions of the mucosa, it was important to restrict the spread of current at each of the stimulation sites. If current spread were sufficiently great, it might affect more than one of the test nerve branches and result in a bias against f inding units that responded selectively to only one of the nerve branches. To prevent significant spread of stimulus current, several steps were taken. These included the use of a stimulus isolation unit , the use of a very short pulse duration (0.1 ms), and the selection of nerve branches that were widely separated

from each other. In order to test the effectiveness of these precautions, control experiments, which

involved testing for bulbar unit responses before and after cutting one of the test nerve branches, were conducted in two different preparations (see Fig. 2 for details). The results of these control experiments demonstrated that if there were any spread of stimulus current , this current was incapable of producing a bulbar unit response via

neighboring nerve branches.

Electrode Placement In order to identify the regions of the olfactory bulb having the highest density of mitral cells, the olfactory bulbs of five animals were prepared for histological examination.

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COSTANZO ANn MOZELL Projection of Nasal Mucosa onto Olfactory Bulb 301

k_ I GROUND ELECTRODE

÷

OLFACTORY BULBS

OLFACTORY SAC

INTERNAL NARIS

EXTERNAL NARIS

FIGURE 1. Diagram of the prepara t ion showing the olfactory nerve branches lifted onto the st imulating electrodes and the recording microelectrode posi t ioned in the olfactory bulb. The inset shows a response typical of those recorded f rom a single unit in the mitral cell layer of the olfactory bulb after the electrical stimulation of an olfactory nerve branch. Vertical bar, 1 mV; horizontal bar, 100 ms.

Sagittal sections, horizontal sections, and cross sections were all s tudied, and as can be seen in Fig. 3a , the olfactory bulb of the frog was found to be organized into four histologically distinct layers. As seen from the surface, the mitral cells were found to occupy a more central region of the olfactory bulb (see Fig. 6) falling within the (x,y) coordinate positions (-+8, -+4).

Electrode penetra t ions were made within those coordinate positions found to contain the highest density of mitral cells. In five di f ferent animals, histology was used to confirm that the electrode had been located within the mitral cell layer; In these five prepara t ions , after recording from a single unit, the tapered shaft o f the microelectrode was p inched off at the surface of the bulb and the location of the remaining tip was identif ied in the histological sections. In all but one o f these preparat ions each electrode tip was clearly located within the mitral cell layer (see Fig. 3b). In the one remaining prepara t ion , even though the electrode tip itself could not be identif ied, the position o f the electrode shaft strongly indicated that it had pene t ra ted into the mitral cell layer.

R E S U L T S

Nerve Branch Response Profiles for Bulbar Units

T h e n e r v e b r a n c h r e s p o n s e p r o f i l e o f a un i t was c h a r a c t e r i z e d f r o m its p a t t e r n o f r e s p o n s e s o r l ack o f r e s p o n s e s to t h e s t i m u l a t i o n o f e ach o f t he t h r e e n e r v e b r a n c h e s . I f a un i t r e s p o n d e d to t he s t i m u l a t i o n o f a g iven n e r v e b r a n c h at a n y

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o f the test vo l t ages (10-60 V), it was c o n s i d e r e d to have a pos i t ive r e s p o n s e to tha t n e r v e b r a n c h .

T h e re su l t s o f a typ ica l e x p e r i m e n t u s i n g the a b o v e c r i t e r i a a r e i l l u s t r a t e d d i a g r a m m a t i c a l l y in Fig. 4. T h e un i t s t u d i e d was l oc a t e d at a c o o r d i n a t e p o s i t i o n ( + 5 , +2) wh ich is a r e l a t ive ly l a t e r a l p o s i t i o n in the o l f a c t o r y bu lb . T h i s un i t was c h a r a c t e r i z e d as h a v i n g a "0+ + " n e r v e b r a n c h r e s p o n s e p r o f i l e , i n d i c a t i n g t ha t it d i d n o t r e s p o n d to s t i m u l a t i o n o f t he m e d i a l n e r v e b r a n c h b u t d i d r e s p o n d to s t i m u l a t i o n o f t he i n t e r m e d i a t e a n d l a t e r a l n e r v e b r a n c h e s . A l t h o u g h in Fig . 4 the r e s p o n s i v e n e s s to on ly o n e se r ies o f tes t vo l t ages is s h o w n fo r e ach n e r v e b r a n c h , in m o s t e x p e r i m e n t s ( i n c l u d i n g t ha t i l l u s t r a t e d in Fig . 4) the e n t i r e s t i m u l u s ser ies was r e p e a t e d at leas t two t imes .

I 2 3 4

FIGURE ~. Control exper iment for the spread of stimulus current . Figure on the left illustrates the responsiveness of a bulbar unit to the stimulation of two neighboring nerve branches in the olfactory mucosa. Once the responsiveness of the unit (frames 1 and 2) to stimulation of the two adjacent nerve branch sites (A and B) was confirmed, one of the nerve branches was carefully severed at a point between the stimulation site (B) on the distal port ion of the nerve and the port ion of the nerve more proximal to the olfactory bulb (see figure on right). After cutt ing the nerve, stimulation to the distal nerve s tump (site B) was found to no longer elicit a response (frame 4). This indicated that there was no spread of stimulus current from site B sufficient to give a response in neighbor ing nerve branches. The response to subsequent stimulation of the uncut branch (frame 3) served as a control to assure that the bulbar unit had not been lost when severing the nerve branch. The position of the single unit in the olfactory bulb is indicated by the filled circle. Arrows indicate stimulus onset. Each record is 500 ms in durat ion.

Fig . 5 i l lus t r a t e s t he r e s p o n s e p r o f i l e s o f t h r e e a d d i t i o n a l uni t s . T h e s e t h r e e uni t s a r e all f r o m the s a m e a n i m a l a n d a p p e a r f r o m lef t to r i g h t in the o r d e r in. wh ich t h e y w e r e e n c o u n t e r e d . T h e t h r e e r e p l i c a t i o n s o f t h e s t i m u l u s se r ies fo r each n e r v e b r a n c h s h o w n in Fig . 5 d e m o n s t r a t e tha t r e s p o n s e s e q u e n c e s we re qu i t e r e p r o d u c i b l e .

T h e f i rs t u n i t (Fig. 5) was l o c a t e d in a m o r e m e d i a l r e g i o n o f the b u l b ( - 8 , +2) a n d was r e s p o n s i v e to on ly t he m e d i a l n e r v e b r a n c h (+00 uni t ) . T h e s e c o n d un i t l oca t ed n e a r t he c e n t e r o f the b u l b ( - 2 , - 2 ) was r e s p o n s i v e to each o f the t h r e e n e r v e b r a n c h e s (+ + + un i t ) . T h e t h i r d un i t , l o c a t e d in a m e d i a l r e g i o n o f t he

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FIGURE 3. A, Sagittal secdon of the olfactory bulb illustrating the principal cell layers. Hematoxylin and eosin stain. Horizontal bar, 650/xm. B, Sagittal section of the olfactory bulb showing the electrode tip located in the mitral cell layer. Cresyl violet stain. Horizontal bar, 200/xm. N, nerve layer; Gl, glomerular layer; M, mitral cell layer; Gr, granular cell layer.

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bulb ( - 4 , - 3 ) , was responsive to bo th the medial and in te rmedia te but not the lateral nerve b ranch (+ +0 unit).

In addi t ion to the nerve b ranch response profiles i l lustrated in Figs. 4 and 5, three o ther response profiles were observed , giving a total o f seven d i f fe ren t response profiles. T o summar ize , they were: (a) units that were dr iven by st imulation o f the medial nerve b ranch but not by the in te rmedia te or the lateral nerve branches (+00 units); (b) units that were dr iven by the medial and in te rmedia te ne rve branches but not by the lateral nerve b ranch (+ +0 units); (c) units that were dr iven by the medial , in te rmedia te , and lateral nerve b ranches (+ + + units); (d) units that were dr iven by the lateral but not the in te rmedia te or the medial ne rve branches (00+ units); (e) units that were dr iven by the lateral and in te rmedia te nerve b ranches but not the medial ne rve b ranch (0+ + units); (/c) units that were dr iven by the in te rmedia te nerve b r anch but not the media l or

~ (+5,+2)

A B C /

VOLTS A B C I0 o o o 20 o o + 30 o o +

4 0 o + +

5 0 o + + 6 0 o + +

FIGURE 4. Diagrammatic representation of a typical experiment. Figure illustrates the left olfactory bulb with coordinates indicating the position of the bulbar unit (()). A, B, and C represent the most medial, the intermediate, and the most lateral olfactory nerve branches respectively, as they are lifted onto stimulating hook electrodes. Table lists the responses to stimulation of the respective nerve branches at the voltages listed. This unit has a 0+ + nerve branch response profile.

the lateral nerve b ranches (0+0 units); (g) units not dr iven by any o f the three nerve branches (000 units). One addi t ional type, + 0 + , was theoretically possible, but was not f ound in the 93 units s tudied.

It is conceivable that d i f ferences in e lectrode impedance or d i f ferences in the contact o f the electrodes with their respective nerve b ranches could account for the various response profi les observed. However , both the techniques emp loyed and the results observed make this possibility a p p e a r highly unlikely. First, the 250-/zm p la t inum wire hook electrodes used for s t imulat ion had relatively low impedance and p rov ided a relatively large contact a rea with the nerve branches . Second, a wide r ange of voltages was used to st imulate each nerve b ranch and if a butbar unit r e s p o n d e d to any o f these voltages, that nerve b ranch was, wi thout fu r the r qualification, simply classified as having an exci tatory effect. Finally, as exempl i f ied in Fig. 5, units with very d i f fe ren t response profi les were quite of ten

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observed even when, in the same p repa ra t ion , the position o f the s t imulat ing electrodes r e m a i n e d una l te red . I n d e e d , units with d i f fe ren t response profi les were found in 71% of the p repa ra t ions in which more than one unit was tested and in which the e lect rode posit ions r ema ined stat ionary. Such results do not seem c o m m e n s u r a t e with the suggest ion that e i ther e lec t rode i m p e d a n c e or the e lectrode contact pe r se can under l ie the response profi les observed for di f ferent bulbar units.

+ 0 0 + + + + + 0

l -S,+2) I - 2 , - Z ) (--4,-31

A B C A B C A B C

VOLTS A B C A B C A B C

lO 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 20 + + + 0 0 0 0 0 0 + 0 0 + + + 0 0 0 0 0 0 0 0 0 0 0 0 30 + + + 0 0 0 0 0 0 + + + + + + + + + 0 0 0 0 0 0 0 0 0 40 + + + 0 0 0 0 0 0 + + + + + + + + + + + + + 0 0 0 0 0 50 + + + 0 0 0 0 0 0 + + 4 + + + + + + + + + + + + 0 0 0 60 + + + 0 0 0 0 0 0 + + + + + + + + + + + + + + + 0 0 0

FIGURE 5. Responses of three bulbar units illustrating three different nerve branch res[5onse profiles. Each unit is from a different bulbar position in the same preparation. Lower tables illustrate responses to nerve branch stimulation. Re- sponses to three different replications of each stimulus voltage for each nerve branch are given. Above the diagram of each olfactory bulb is given the nerve branch response profile for the respective unit. The meaning of the different symbols used to depict the coordinate positions of the units will be discussed in conjunction with Fig. 6.

Topographical Distribution of Units with Different Nerve Branch Response Profiles

Fig. 6 shows the results o f 51 single units sampled f r o m di f fe ren t locations across t h e olfactory bulb. T h e olfactory bulb was cons idered to be divided into three genera l regions: a medial reg ion , an in te rmedia te region, and a lateral region. 17 units f r o m each o f these th ree regions (51 in all) were classifed accord ing to their nerve b ranch response profiles.

Units which did not r e s pond to s t imulat ion to any o f the three nerve b ranches (000, open circles) were dis t r ibuted nonspecifically across the d i f fe ren t regions o f the ol factory bulb. Units responsive to each o f the th ree ne rve b ranches (4-,+ + , filled circles) were similarly dis t r ibuted nonspecifically.

O f par t icular interest were those units which f r o m their response profi les a p p e a r to receive their exci tatory inputs f rom specific regions o f the ol factory mucosa. For ekample , the response profi les o f some units (+ +0 and +00 units)

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0 0 t ~ t t • ~

T H E J O U R N A L OF G E N E R A L P H Y S I O L O G Y • V O L U M E 6 8 • 1 9 7 6 306

~ 0 0 0

Q

~ m I I 0 + 0 1 1 ~ - ~ I I I

o 9 ~ • o

O

0 0 0 0 + o o

qD + + 0 • + + +

0 + + 0 0 + o + o

FIGURE 6. Topographica l distribution of 51 bulbar units in relation to their olfactory nerve branch response profiles. The position of each unit on the bulbar coordinate system is shown and the symbols used to designate the response profile of each unit are given in the inset. To the right of each symbol in the inset is the nerve branch response profile which it represents. The zeros (O's) and pluses (+'s) in each response profile indicate whether or not the respective nerve branches shown in the inset excited the unit. Example, filled circles, O, show the positions of those units having a + + + response profile meaning that they respond to electrical stimulation of each of the three nerve branches. Each division on the grid represents approximate ly 100/zm.

i n d i c a t e d a bias to i n p u t s f r o m the m o r e m e d i a l a s p e c t o f t he m u c o s a . T o i l lus t ra te this m e d i a l b ias in Fig . 6 t hese un i t s a r e d e s i g n a t e d by ci rc les s h a d e d on the le f t ( i .e. s h a d e d on the m e d i a l s ide) . As can be seen in Fig . 6, all un i t s h a v i n g a m e d i a l bias w e r e l oca t ed on ly in t he mos t m e d i a l r e g i o n o f the o l f ac to ry b u l b . N o un i t s o f th is t y p e we re f o u n d in t he i n t e r m e d i a t e o r l a t e r a l r e g i o n s o f t h e

b u l b . O t h e r un i t s we re f o u n d to have a bias to i n p u t s f r o m the m o r e l a t e r a l a s p e c t o f

t he m u c o s a a n d were t h e r e f o r e d e s i g n a t e d in Fig . 6 by ci rc les s h a d e d o n the

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right (lateral side). These laterally biased units were found only in the lateral region of the bulb.

One unit was found to have a selective input f rom the intermediate nerve branch alone. This unit was located in an intermediate region of the olfactory bulb and is designated by a starred circle with a bar down the center.

Columns A - E of Table I summarize the data shown in Fig. 6. For each of the three regions o f the bulb is given the actual n u m b e r o f cells within each response profile category. This n u m b e r is also given as a percentage o f the 17 cells sampled in each of the three bulbar regions. Cells having a medial bias (+00 and + + 0 units) represent 35% of the cells sampled f rom the medial bulb. Laterally biased cells (00+ and 0 + + ) made up 47% of those sampled f rom the lateral bulbar region. ( If one were to eliminate f rom the analysis the 000 cells that did not respond at all to any of the three nerve branches, the percentage o f cells showing a medial or lateral bias would, o f course, be even greater.)

Increased Sample Size

The results o f the experiments given in Table I ( A - E ) show that a considerable propor t ion o f the cells in different regions of the olfactory bulb have a bias with respect to inputs f rom different regions o f the olfactory mucosa. Al though a total o f 51 units were sampled f rom across the olfactory bulb, the number of cells sampled in any one region was relatively small (n = 17) and since there was a possibility o f seven different response profiles that could possibly be part i t ioned a m o n g these 17 units, estimates o f the percentages o f each type o f response profile could be considered first approximat ions only. Fu r the rmore , the failure to observe cells with particular types o f response profiles in a given region of the bulb (for example, a laterally biased unit in the medial bulb) may have also been the result o f this small sample size. To most efficiently increase the sample size in o rder to determine whether units with a particular mucosal regional bias are restricted to a part icular bulbar region, an additional 42 units were sampled f rom the lateral region o f the bulb only. This increased the total n u m b e r of cells

T A B L E I

NUMBER OF CELLS (AND PERCENTAGES) IN EACH RESPONSE PROFILE CATEGORY FOUND IN THE THREE REGIONS OF THE OLFACTORY BULB

B C D E A Response profile Medial Intermediate Lateral

n = 1 7 % n = 1 7 % n = 1 7

Medial bias +00 5 29 0 0 0 0 ++0 1 6 0 0 0 0

Intermediate bias 0+0 0 0 1 6 0 0

Lateral bias 00+ 0 0 0 0 3 18 0++ 0 0 0 0 5 29

No bias + + + 5 29 8 47 4 24

No response 000 6 35 8 47 5 29

F Lateral

% n = 5 9

0 0

0

11 13

22

13

%

0 0

0

19 22

37

22

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3 0 8 T I l E J O U R N A L OF G E N E R A L P H Y S I O L O G Y " V O L U M ~ 6 8 " 1976

sampled from this region to 59 units. Fig. 7 shows the locations in the olfactory bulb of all 59 o f these units.

Column F of Table I summarizes the results from the 59 units which can be compared to the data for the original 17 unit sample given in column E. Even with the increase in sample size, the relative percentages of the different cell types C~cl not change appreciably. Fur thermore, those response profiles (+00, + +0, 04-'0) that were not present in the small lateral cell sample did not appear when the i~"h~fnber of cells sampled was increased threefold.

D I s c u s s I O N

Topographical Projections

Fig. 6 illustrates the existence of a topographical organization of neural projections from different regions of the receptor sheet onto different regions o f the olfactory bulb. Cells in the medial region of the olfactory bulb are frequently

Q ~ 0 ¢ •

o ~ • * 8

FIGURE 7. The response profiles of 59 units sampled from the most lateral region of the bulb. The coordinates and the symbols are the same as in Fig. 6. The boxed- in area calls attention to the most lateral of the three bulbar regions.

biased to inputs from the medial region of the mucosa and cells in the lateral region of the bulb are frequently biased to inputs from the lateral regions of the mucosa. Furthermore, as seen in Table I, no instances of crossover in these projections were found. That is, there were no medially biased cells (+00, + +0) in the lateral region of the bulb and no laterally biased cells (00+, 0+ +) in the medial region of the bulb, These findings give functional support to the studies of Le Gros Clark (1951) and those of Land (1973) which provided histological evidence for topographical projections. Fur thermore, these findings reveal an organization of projections which is readily compatible with what might be ex- pected if olfactory discrimination were based upon a spatiotemporal analysis (Adrian, 1954).

Spatiotemporal Analysis of Odorants As already indicated, there are at least two different but not mutually exclusive hypotheses for the mechanisms generating spatial and temporal patterns: (a)

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clusters (or aggregates) of selectively sensitive receptors, and (b) differences in the distribution of odorant molecules across the mucosa. In either case, the analysis made at the mucosal level should be preserved for fur ther processing in the more central levels of the olfactory system by a topographical projection of the mucosa onto the olfactory bulb.

Consider the case in which the olfactory receptor sheet consists of clusters of receptor cells having different degrees of sensitivity to different odorants. Consider fur ther that clusters of cells with similar sensitivities may be spaced across the mucosa in different sequential locations. Each odorant would then give rise to a unique spatial and temporal pattern of activity as its molecules, moving along the mucosa, interact with its particular sequence of sensitive clusters. I f such selectively sensitive clusters are present in the mucosa, the spatiotemporal pattern that they would establish could be preserved for the central nervous system by the presence of topographical projections such as those observed in this study.

However, it should be noted that these clusters, if they do exist, might be arranged in a finer mosaic than could be sampled by the size of the nerve bundles stimulated in the present study. Therefore , to fur ther investigate these putative clusters, the techniques employed might have to be refined to allow for the electrical stimulation of nerve branches more peripherally where they are subdivided into even smaller fascicles. Consequently, in the strong support given by this study to the general concept of a topographical projection of mucosal activity onto the bulb, no suggestion is made to preclude the possibility that an even finer point-to-point projection might exist than that reported here.

In addition to clusters of selectively sensitive receptors, spatiotemporal differ- entiation might be based upon the different migration patterns of various odorants across the mucosal sheet. That molecules of different odorants move across the mucosa in different space-time distribution patterns has been strongly indicated by both electrophysiological and gas chromatographic studies (Mozell, 1966, 1970; Mozell and Jagodowicz, 1973). In addition, recent isotope studies have shown a steep concentration gradient from the external naris to the internal naris after a "sniff ' of tritiated butanol molecules (Hornung et al., 1975). Such differential distributions of molecules across the mucosa could be reflected in the activity of the olfactory bulb by means of a topographical projection of the mucosa onto the bulb.

Consider for an example two chemicals, carvone and octane, known to produce different activity patterns across the olfactory mucosa (Mozell, 1970). Carvone gives a response more limited to the medial region of the mucosa than does octane. The latter gives responses of about equal strength in both the lateral and the medial regions. Conceivably, then, both chemicals might excite those cells in the medial bulb which have a bias to the medial mucosa. However, those cells in the lateral bulb having a bias to the lateral mucosa might respond more strongly to octane than to carvone. Thus, in terms of the present results, octane might excite all those cells in the bulb having the following response profiles: 00+, 0+ +, 0+0, + +0, +00, + + +. Carvone, on the other hand, would excite most those bulbar cells with the following response profiles: +00, + +0, + + + . Therefore , these two chemicals would initially establish two different

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3 1 0 THE .JOURNAL OF GENERAL PHYSIOLOGY'VOLUME 6 8 " 1976

activity pat terns across the mucosa. A reflection of these pat terns could then be preserved across the bulb by the topographical project ion functionally demonstrated in the present study.

Inhibition

Although a "0" in the response profile indicated the absence o f an excitatory influence o f a part icular nerve branch on a bulbar unit , it did not necessarily indicate that there was no inf luence at all f rom that nerve branch. It is possible that the bulbar unit may have been inhibited ra the r than simply not excited. I f this were the case, a 0 in a unit's response profi le could represen t an inhibitory region in its receptive field. T h e exper imenta l design was not in tended to distinguish between these two alternatives and indeed the low rate o f sponta- neous activity (circa 0.4 spikes/s) for the bulbar units would make such observa- tions particularly tenuous. At any rate, the possibility of inhibitory regions in the receptive field o f a bulbar cell is impor tan t since in o ther sensory systems (Mountcastle, 1957; Hubel and Wiesel, 1968; Hart l ine et al., 1956; Rose et al., 1959) lateral inhibition in part icular has been found to be fundamenta l to the sharpening o f stimulus differences. Perhaps a similar mechanism occurs in olfaction.

Receptive Field Size

T h e response profiles o f a bulbar unit (Figs. 6 and 7) can be taken as an index of the size of its excitatory receptive field across the ro o f o f the olfactory sac. Some units (viz., +00, 0+0, 00+) appea r to get their excitatory inputs f rom relatively small regions o f the dorsal olfactory mucosa. Other cells ( + + 0 and 0+ +) have wider projections and still o ther cells ( + + + ) did not appea r to have any localization of inputs f rom the dorsal mucosa. Thus it appears that the overall populat ion o f second o rde r cells in the olfactory bulb is made up o f d i f ferent cell types each having d i f fe ren t degrees o f spatial specificity f rom the receptor sheet. However , it should be noted that since this study was conf ined to nerve branches subserving the dorsal recep tor sheet, the size of the receptive fields o f some bulbar units may have been somewhat underes t imated .

Recognizing the possibility o f species variations in the precision of the projection onto the bulb o f part icular epithelial regions, it might still be pointed out that the dif ferences in receptive field sizes r epor ted here are not inconsistent with the results of the earlier histological work cited above. This histological work also showed mucosal projections onto the rabbit bulb which varied widely along a con t inuum runn ing f rom "precise" to "diffuse." In addit ion, the present findings do not seem inconsistent with those o f F reeman (1974). His observation that the stimulation of olfactory nerve axons produces an evoked potential encompassing much o f the bulbar surface seems compatible with the present observation that each part o f the bulb has many units which receive their inputs f rom wide regions o f the mucosa. Perhaps the spatial shift in the epicenters o f the evoked potentials which Freeman noted as he changed the position o f his stimulating electrode reflects the change that this maneuver would en g en d e r in the pool of those bulbar units (identified in the present study) which receive their excitation f rom more localized receptive fields.

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COSTANZO AND MOZZLL Projection of Nasal Mucosa onto Olfactory Bulb 311

Implications

O t h e r s e n s o r y sys tems h a v e b e e n s h o w n to p r o j e c t t he spa t i a l o r g a n i z a t i o n o f t h e i r p e r i p h e r a l r e c e p t o r s h e e t o n t o m o r e c e n t r a l levels o f t he sys tem. I n s o m e cases th is p r o j e c t i o n c o u p l e d wi th l a t e r a l i n h i b i t o r y i n t e r a c t i o n s is cr i t ica l to t he analys is o f t he s t i m u l u s s p e c t r u m which beg in s in t h e p e r i p h e r y as a spa t i a l d i s t r i b u t i o n ac ross a r e c e p t o r shee t . T h i s s t u d y has f u n c t i o n a l l y d e m o n s t r a t e d a t o p o g r a p h i c p r o j e c t i o n o f the o l f a c t o r y r e c e p t o r shee t o n t o its s e c o n d o r d e r n e u r o n s in t he o l f a c t o r y b u l b . I t m a y be tha t , as in o t h e r s e n s o r y sys t ems , a spa t i a l ana lys i s o f t he s t i m u l u s at t he r e c e p t o r level is a n essen t i a l f e a t u r e in t h e

p r o c e s s i n g o f o l f a c t o r y i n f o r m a t i o n .

The authors with to thank Dr. R. J. O'Connell of The Rockefeller University for his helpful review of the manuscript. This research was supported by the National Institutes of Health Research Grant No. NS 03904 and the National Institutes of Health Training Grant No. ST 01G M1006.

Received for publication 9 February 1976.

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