Olfactory pathways and the sense of smell

Neuroscience and Biobehavioral Reviews, Vol. 16, pp. 453-472, 1992 0149-7634/92 $5.00 + .00 Printed in the U.S.A. All rights reserved. Copyright © 1992 Pergamon Press Ltd.

Olfactory Pathways and the Sense of Smell

B U R T O N M. S L O T N I C K .1 A N D F R A N C E S W. S C H O O N O V E R t

*Department o f Psychology, The American University, Washington, DC 20016 t Institute o f Psychiatry and Human Behavior, University o f Maryland Medical System, Baltimore, MD 21201

Received 22 July 1991

SLOTNICK, B. M. AND F. W. SCHOONOVER. Olfactory pathways and the sense of smell. NEUROSCI BIOBE- HAV REV 16(4) 453-472, 1992.-Rats were trained using operant conditioning to detect isoamyl acetate vapor generated by an olfactometer. They received lesions of olfactory pathways and were tested for retention of the odor detection task and trained on two-odor tasks. Deficits in odor detection and two-odor discrimination were related to the extent to which lesions disconnected the olfactory bulb from the forebrain. Transection of only the lateral olfactory tract, only the anterior limb of the anterior commissure, or lesions of the olfactory tubercle had little effect but combined lesions of these structures produced severe deficits in both odor detection and discrimination. Only rats with almost complete transection of the olfactory peduncle or cortex were anosmic; those with transections that spared a small segment of tissue between the olfactory bulb and olfactory cortex had detectable olfactory function. The results are discussed with regard to efferent connections of the olfactory bulb.

Olfactory bulb Olfactory cortex Lateral olfactory tract Anterior commissure Anosmia Odor detection Odor discrimination

THE connections of the olfactory bulb with the forebrain and brainstem have been described in detail but few studies have examined the effects of transecting these pathways on the ability to smell. Most prior attempts to identify brain areas significant for olfaction have yielded largely negative results. In the classic studies of Allen with dogs (2,3) and Swann with rats (69) extensive lesions of the neocortex, destruction of most of the septum, amygdala, piriform lobe, and complete extirpa- tion of the hippocampus were largely without effect on learned olfactory discriminations. In Allen's studies, dogs with removal of the piriform lobe and amygdala, or with frontal lobectomy, could perform a simple odor detection task but were unable to inhibit responding to a negative conditioned odor. This deficit was probably related to the now well-known inability of animals with frontal cortical or amygdala lesions to inhibit responding in a variety of "go, no-go" tasks (6,57), and not to an inability to discriminate odors. Subsequent studies have failed to find deficits in olfactory discrimination after lesions or olfactory deafferentation of the amygdala [e.g., (9,10,55,59)].

Several studies have demonstrated deficits in olfactory cued behavior after destruction of the mediodorsal nucleus in the rat (18,32,64,66,68). Such lesions do not result in anosmia or, according to Eichenbaum, Shedlack, and Eckmann (18), even a change in olfactory sensitivity but, instead, may alter the animal's ability to acquire difficult or complex odor discrimination tasks.

Clear deficits in sensory capacity have been obtained only after removal of the olfactory bulbs or, in some cases, after transection of primary olfactory tracts. Bilateral olfactory bulbectomy uniformly results in severe deficits in tests of odor

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453

detection or discrimination and is often used as a control procedure to assess the effects of other central lesions on conditioned and species-specific odor-cued behavior and to determine the degree to which a behavior is olfactory-dependent [see (39) for review].

The extent to which transection of olfactory pathways du- plicates the deficits produced by olfactory bulbectomy is less clear. In Swann's study, three rats had lesions of olfactory tracts. One, with bilateral destruction of the lateral olfactory tract (LOT) and unilateral transection of the anterior limb of the anterior commissure (AC) had only a moderate deficit in odor discrimination. The other two had bilateral destruction of LOT plus bilateral damage to the AC and had much more severe deficits. Based on these results Swann suggested that the anterior commissure rather than the LOT may be the critical pathway for olfaction. However, this conclusion has not been supported. Brown and Geselli (9) and Long and Tapp (31) found no deficits in simple odor discrimination after bilateral interruption of the AC, although Bennett (5), using sensitive psychophysical tests, showed that such lesions produced moderate deficits in retention and sensitivity.

Reports on the effects of transecting the LOT also are not entirely consistent. Long and Tapp found that the performance of rats with lesions of the LOT tested on olfactory discrimination tasks was similar to that of olfactory bulbectomized rats, and Thompson (70) found that rats with LOT transections, like those with bilateral bulbectomy, had no retention of a preoperatively learned odor detection problem. Other evidence that transection of the LOT may produce anosmia or very severe deficits in olfaction comes from studies of maternal and sex behavior. Fleming and Rosenblatt (20)

454 SLOTNICK AND SCHOONOVER

found that changes in maternal behavior produced by olfactory bulbectomy could be duplicated by lesions of the LOT and Devor (13) reported that hamsters with either olfactory bulbectomy or with anterior transection of the LOT showed no sex behavior. Similar deficits in hamster sex behavior from both anterior and posterior transection of the LOT have been reported by Marques, O'Connell, Benimoff, and Marcrides (36) and most experimental animals in this study failed in a simple test of olfaction (localizing a food odor).

In contrast, several studies showed that rats with complete transection of the LOT are not anosmic. Gervais and Pager (24) found that rats with unilateral transection of the olfactory peduncle and contralateral transection of the LOT showed only a transitory deficit in a food seeking task. Slotnick and Berman (61) reported that discrete transection of the LOT was largely without effect on an odor detection task in one rat. More severe deficits were obtained in rats with large lesions of the anterior olfactory nucleus (AON) that included the LOT but these animals were not anosmic and their discrimination performance improved with training. Cattarelli (11), using both electrophysiology and behavioral measure (reactivity to odors), found that rats with bilateral transection of the LOT had a severe sensory deficit but were not anosmic.

In summary, it appears that destruction of a variety of neocortical and limbic structures may be largely without effect on simple tests of olfaction and that only lesions of the princi- pal olfactory pathways (and of the olfactory bulbs) produce marked behavioral deficits in the detection and discrimination of odors. No study has identified a rhinencephalic pathway or locus (other than the olfactory bulb) that is essential for odor detection.

SOME ANATOMICAL CONSIDERATIONS

These outcomes are, perhaps, not unexpected because discrete lesions or transection of a single pathway would spare important connections between the forebrain and olfactory bulb. At least 3 direct efferent bulbar pathways to olfactory cortex have been identified and, in addition, several indirect pathways connect the olfactory bulb to olfactory cortex. The direct pathways include the lateral olfactory tract and the more diffuse or less well-defined medial fiber systems project- ing to the olfactory tubercle and to the tenia tecta (TT).

The Lateral Olfactory Tract

The extent to which transection of the lateral olfactory tract disrupts bulbar input to the olfactory peduncle and cortex depends significantly upon the level at which it is interrupted. The tract forms at the caudal aspect of the olfactory bulb where efferent axons collect in a dense bundle that bounds all but the medial aspect of the olfactory peduncle. It is difficult to completely transect the tract at this rostral level without producing significant damage to the AON and its fiber systems. Thus, in prior behavioral studies the tract had been cut at a caudal level where it is more compact. However, transection of the LOT, even at the posterior edge of the olfactory peduncle, may leave intact connections from the LOT to the AON, as all parts of the AON receive an input from the LOT (7,15,41,43,49,51,53,54,73). Available evidence suggests that this projection to the olfactory peduncle from the LOT consists of main axons (or sustaining collaterals) and, therefore, would not be disrupted by a posterior LOT transection. Thus, Price and Sprich (47) found that many LOT axons terminate in the rostral olfactory cortex and, while the separate contribution of LOT fibers to the AON was not

specifically determined, Friedman and Price (23) reported that transection of the LOT caudal to the AON did not disrupt the normal termination of LOT axons in the AON. Thus, transection of the LOT at or posterior to the caudal aspect of the peduncle does not deprive the more rostral AON nuclei of bulbar connections and, as described below, projections from these nuclei provide input to the olfactory cortex.

Medial Olfactory Tracts

Bulbar efferents that are not part of the main or compact part of the LOT include those to the tenia tecta and to the olfactory tubercle. In the rat, the ventral, but not the dorsal, part of the tenia tecta receives a direct input from the olfactory bulb.

The bulbar projection to the olfactory tubercle originates, in part, from middle and external tufted cells (52). The pathway appears to travel in the medial half of the peduncle and may originate from the medio-caudal part of the olfactory bulb (15). Of particular relevance to the present study are Devor's experiments demonstrating anterograde degeneration of bulbar efferents after olfactory bulbectomy in hamsters, which had a prior transection of the LOT (15,16). The degeneration produced by the bulbectomy revealed which projections to olfactory cortex were spared by the prior LOT transection. Complete transection of the LOT or transection of the medial half of the LOT at the caudal aspect of the peduncle did not denervate the olfactory tubercle or the medial wall of the peduncle caudal to the cut. A bulbar projection to the olfactory tubercle in hamsters with transection of the LOT has also been demonstrated using autoradiography by Marques, O'Connell, Benimoff, and Marcrides (36).

Indirect Connections

In addition to these direct bulbar projections, the olfactory cortex also receives an input from those components of the olfactory peduncle that are directly connected to the bulb: the AON, the ventral tenia tecta and the dorsal peduncular cortex. The olfactory cortical projections from the AON travel in, or are associated with, the anterior limb of the anterior commissure. While this commissure is generally considered a crossed pathway by which olfactory impulses from the AON influence the contralateral AON and olfactory bulb, a significant number of axons leave the anterior limb of the tract and terminate in the piriform cortex and olfactory tubercle both ipsilateral and contralateral to the cells of origin. This projection, first reported by Powell, Cowan, and Raisman (42) in the rat, has subsequently been described in somewhat greater detail in the rabbit (8) and mouse (Slotnick and MacLean, unpublished observations). Axons leave the anterior limb of the anterior commissure in a postero-ventral-lateral direction and appeared to terminate in layer III, II and Ib of the piriform cortex and olfactory tubercle (8). Thus, this projection provides an input to the piriform cortex that is separate from that of the LOT. The laminar pattern of the AON projections to olfactory cortex are complementary to that of the direct projections from the olfactory bulb. The direct projections (from the LOT) terminate in layer I a while the indirect projections (from the AON) terminate in layer lb.

In addition, the dorsal peduncular cortex, a discrete area on the dorsal aspect of the olfactory peduncle, which is transi- tional between the AON and piriform cortex, receives a direct input from the bulb and projects to the entorhinai cortex, piriform cortex and olfactory tubercle (27). It is unclear

OLFACTORY PATHWAYS AND THE SENSE OF SMELL 455

whether these latter projections travel in the anterior commissure.

Finally, the ventral tenia tecta also projects to piriform cortex and olfactory tubercle. [Interestingly, in the mouse, both divisions of the tenia tecta receive a direct projection from the bulb and the dorsal tenia tecta, which is continuous with the anterior hippocampai rudiment, provides a direct olfactory input to the hippocampal formation (1,54). Other species differences in olfactory connections probably exist, e.g., (1,4,53,54), and must be considered in assessing the effects of lesions on olfaction].

As shown by Haberly and Price (26,27) and Luskin and Price (33,34), the projections of the olfactory peduncle are part of a complex association system that interconnects all parts of primary olfactory cortex and includes extrinsic projections from the cortex to the diencephalon (26,44,45). The projections originating from the AON are of particular interest in the present study because they could provide a path for olfactory impulses into both the ipsilateral and contralateral olfactory cortex (and diencephalon) in animals with transection of the main body of the LOT.

In summary, there are multiple pathways for the projection of olfactory impulses from the olfactory bulb to the forebrain. Further, the projections to olfactory cortex appear to be relatively diffuse, such that any one area of cortex may receive input from different regions of the olfactory bulb and olfactory peduncle [e.g., (33,50,51)]. The limited success of prior studies in producing frank anosmia or a severe hyposmia from relatively large forebrain lesions or transection of primary olfactory pathways might suggest either that large areas of the brain are equipotential for making olfactory discriminations, or that information processing is occurring at relatively rostral portions of the olfactory system. But, such conjectures are certainly premature because there has been no systematic study of functional olfactory neurology or of the relative contribution of different projection pathways to even simple odor tasks, such as detection and two-odor discrimination. Further, the test procedures used in most prior studies were inadequate to accurately assess the nature and extent of a sensory loss, were probably insensitive to small changes in sensory capacity, and provided little control of stimulus intensity and odor sampling behavior of subjects (60). In addition, the behavioral measures, methods of odor delivery, odorants used as discriminative stimuli and the anatomical presentation of results varied widely in these studies, thus severely limiting meaningful comparisons among them.

The limited success of the few available studies leaves fun- damental questions concerning the neural basis of olfactory detection and discrimination unanswered. The present study addresses the most basic of these: Which bulbofugal pathways are essential for the detection and discrimination of odors? To this end, rats were given extensive preoperative training on a simple odor detection task and tested for retention and for two-odor discriminations after lesions of primary olfactory pathways.

METHOD

Subjects

Fifty-nine adult male Wistar albino and Long-Evans hooded rats were used. The animals were maintained in a temperature- and humidity-controlled vivarium in individual plastic cages.

Apparatus

A diagrammatic representation of the air-dilution olfactometer used for odor detection and intensity-difference discrimination tests is shown in Fig. 1A. Air, provided by an oil-free compressor regulated at 20 p.s.i., was dehydrated with a refrigerant dryer and filtered through microfiber tubes and a 4 liter column of activated coconut charcoal. The system was constructed entirely of glass and Teflon. The ductwork was 5-mm i.d. glass tubing, and all connections were made with tight-fitting Teflon sleeves. Air flows were controlled by needle valves and monitored by flowmeters. Isoamyl acetate saturated vapor was generated by passing 50 cc/min, of air over 30 ml of isoamyl acetate (Aldrich, >99°7o purity) con- rained in a folded 2.5-cm diameter, 90-cm long glass tube (17). The vapor saturator was submerged in a constant temperature water bath maintained at 19 ° C. Air from the third dilution stage of the system was divided into two 0.3 liters/min stimulus channels, each connected to a separate three-way Teflon

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FIG. 1. Diagrammatic representation of the olfactometers. A, the system used for odor detection and intensity difference discrimination tasks. Air is provided by an oil-free compressor and filtered through silica gel and activated charcoal. Odor saturated air is diluted with clear air in manifolds 1-3 and 6. For odor detection tasks flowmeter C delivered amyl acetate vapor to manifold 5 and flowmeter A delivered clean air to manifold 4. For the odor intensity difference problem, flowmeters A and B were set to deliver a fraction (20%) of the amyl acetate concentration supplied to manifold 5. The animal chamber was connected to a 70 cfm exhaust fan via a flexible hose. See text for additional details. B, the system used for the two-odor discrimination task. Odors were contained in saturators A and B.


solenoid valve. Because each of these stimulus channels was connected in parallel to a clean air channel (Fig. 1A), each could be set to provide odorized or clean air. The normally open ports of each solenoid were connected to an exhaust line via separate flow meters for monitoring exhaust flow and valve operation. The normally closed ports of the solenoids were connected to a manifold (Fig. IA, M6) that received a continuous 2.7-liter/min stream of clean air. The outflow of this manifold was connected to the animal chamber through the common and normally open ports of a three-way Teflon valve (Fig. IA, FV). As described below, operation of this solenoid served to briefly divert the air from the test chamber and to signal the onset of a trial.

For the detection task the olfactometer was set to provide 0.5% (of vapor saturation) amyl acetate through one stimulus channel and clean air through a second channel. For the amyl acetate intensity-difference task, the first channel provided a 0.1% vapor stimulus and the second channel a 0.02% vapor stimulus. The flow rates through the stimulus channels (0.3 liters/min.) and the carrier stream (2.7 liters/min.) were kept constant. All flow meters were calibrated in place using bubble flow meters connected to the downstream end of the system and the flow system was checked for leaks at regular intervals. The system was washed with 95% ethyl alcohol and air dried approximately every 2 weeks during the study. The activated charcoal was renewed by heating at 150 ° C for 12-24 hr every 2 months. Except the vapor saturator, which was isolated by stopcocks when the system was not in use, air flow through the system was never turned off.

A separate air-dilution olfactometer was used for generat- ing two qualitatively different odors. As shown in Figure IB, this unit consisted of two independent dilution systems, each fed by a separate vapor saturator. The over-the-surface saturators were 2.5-cm diameter, 20-cm long glass tubes and were maintained at room temperature.

The animal test chamber consisted of a modified glass funnel attached to a 13-cm-long Plexiglas tube. A perforated glass disc in the stem of the funnel diffused the incoming air stream. A 18-mm long, 6-mm diameter stainless steel bar cemented through the left wall of the funnel served as a response key. Contacts between the bar and a stainless steel floor plate were detected by a touch-sensitive circuit (19). A photocell posi- tioned across the neck of the chamber 2 cm behind the air-diffusing screen served to detect trial-initiating responses. Water reinforcement from a solenoid-controlled reservoir was delivered to a 10-mm diameter glass cup at the base of the funnel. Air from the chamber was passed continuously to the outside of the building by a 70-cfm exhaust fan connected to the chamber by a flexible plastic hose. The test chamber was housed in a refrigerator that was thermostatically controlled to maintain the chamber temperature at 20 ° C.

The operant-conditioning training procedure described below was controlled in the initial stages of the study by a set of solid-state digital modules. In the latter half of the study, control was achieved using an Apple II TM computer and a digital interface.

PREOPERATIVE TRAINING PROCEDURES

Beginning at 14 days prior to training, rats were restricted to 10 ml of water a day. This water deprivation schedule was, in effect, throughout the experiment except for a 3-5 day postoperative recovery period when ad lib water was allowed. The deprivation schedule resulted in stabilization of body weight at 75070-80% of weight prior to deprivation.

Animals were trained to detect and discriminate odors using a discrete trials go, no-go operant conditioning procedure described in detail by Slotnick and Schoonover (67). Briefly, in initial sessions standard operant conditioning procedures were used to train the rat to respond on the bar for a 0.05 ml water reward after initiating a trial by breaking the photobeam with its nose. Each trial-initiating response produced a 5 s presentation of the positive stimulus (0.5°70 isoamyl acetate). By the end of training the following trial parameters and con- tingencies were in effect (Fig. 2). The first photobeam break after a minimum intertrial interval of 5 s initiated a trial. At the beginning of the trial, both the final valve (Fig. 1A, FV) and the stimulus valve operated. This introduced the stimulus into the carrier stream in manifold 6 (Fig. 1A, M6) and, during the initial period in which the two streams mixed, shunted the air to an exhaust path. The final valve was de-energized after 1 s and the odor was diverted into the chamber. Five seconds later the stimulus control solenoid was de-energized (thus, terminating the stimulus-on period). Responses made during the first 2 s of the stimulus-on period (stimulus sampling period of the trial) were ignored. A response of at least 0.3 s duration in the remaining 2 s of the trial (response interval) terminated the trial and produced a 0.5 ml water reward. Any response made while the final valve was operated (when the odor stimulus was not present) immediately terminated the trial and initiated the 5 s inter-trial interval. Trials that terminated with reinforcement were scored as correct (hits). If the animal did not respond during the response interval the trial was scored as an error (miss). Trials in which the animal responded during the final valve period were scored as aborts.

Operation of the final valve for 1 s at the beginning of each trial allowed the stimulus stream to mix with the carrier stream before the stimulus was introduced into the chamber, provided a warning signal for the animal for stimulus presentation, and served to mask any differential sounds of the stimulus control valves. Also, termination of the trial for responses made during the operation of the final valve (when no odor was present) served to promote stimulus sampling. In pilot studies, it was found that stimulus control of behavior was much poorer when the final valve was not used.

The S- stimulus (clean air) was introduced in the next session. Positive and negative trials were presented in random

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FIG. 2. Sequence of events during an S + trial. Upon breaking the photobeam at the end of an intertrial interval the odor and final valve operate. During the l-s final valve period the odor and carrier stream mix and are shunted to an exhaust path (time period A in figure). When the final valve is de-energized the odor is presented to the animal for a maximum of 4 s (time period marked B in figure). Re- sponses made during the first 2 s of the odor-on period were ignored. The first response made in the remaining 2 s was reinforced with 0.05 ml of water and terminated the trial. See text for additional details.


order with the restriction that not more than 3 trials of one type occurred in succession and that there were an equal number of positive and negative trials in each block of 20 trials. Procedures during S- trials were identical with those of S + trials except that S- trial responses were not reinforced. If the animal did not respond during the response interval of a S- trial, the trial was scored as correct (correct rejection). If the animal did respond, the trial was scored as an error (false alarm). Performance accuracy was determined for each block of 20 trials. The outcome of all trials (except aborts) was used in scoring. Criterion performance for all training was set at 90070 correct responding in a block of 20 trials.

Prior to surgery all rats received 600-800 trials (100-200 trials per session) on this detection task (S +, 0.507o isoamyl acetate; S- , air). Generally, each rat was given one session per day. All animals acquired this detection task in the first session and maintained stable performance of 90070-100070 correct responding in the remaining trials. As a control, several rats that were scheduled to receive large lesions of the olfactory cortex were also trained on a visual discrimination. A 24-vdc lamp, located above the air inlet of the chamber, was used to provide a flashing light (S + stimulus) or a steady light (S- stimulus).

Postoperative Tests

The retention test. Eighteen to 21 days after surgery, the rat was tested for postoperative retention of the isoamyi acetate detection task. Once criterion performance of 90°'/0 or higher responding in a block of 20 trials was achieved, the rat was given an additional 200-trial session on the detection task. If the animal did not achieve criterion, performance testing was continued for a maximum of 2000 trials.

The 0.05% isoamyl acetate detection test. In the next session identical test procedures were used except that the concentration of the isoamyl acetate stimulus was reduced to 0.05070. Training was continued until criterion performance was achieved or for a maximum of 600 trials.

The intensity-difference test. In the next session the S + stimulus was 0.1 070 isoamyl acetate and the S- stimulus was 0.02070 isoamyl acetate. Training was continued until criterion performance was achieved or for a maximum of 600 trials.

The two-odor test. In this final test, animals were trained to discriminate between 0.1070 ethyl acetate (S +) and 0.1070 isopropyl acetate (S-). Animals were trained to criterion or for a maximum of 600 trials. Most rats that achieved criterion performance on this task were also tested to determine whether their discrimination was based on the qualitative or quantitative difference between the stimuli. This was done by alternately increasing and decreasing (by approximately a fac- tor of 5) the concentration of both the S + and S- stimuli in blocks of 10 or 20 trials during a regular training session. In these tests the sudden changes of stimulus concentration did not disrupt performance, and it was clear that stimulus control was based on the qualitative difference between stimuli.

Rats that did not achieve criterion performance in the 0.05°70 detection and/or intensity-difference test were re- trained on the 0.5070 detection task before the next test was given. This procedure was adopted to insure that the rat was under stimulus control before it was tested on a new problem.

Not all tests were given to all rats. In the initial stages of the study, some rats were tested only on the 0.5070 detection task and the two-odor task. In a few cases, test results were lost because of equipment failure. Finally, for animals that did not achieve criterion performance in 2000 trials on the

retention test, the more difficult .05070 detection test and the intensity difference test were omitted, but some animals were tested on the two-odor task.

Control Procedures

Preliminary analysis using a flame ionization detector demonstrated that the over-the-surface saturator produced air saturated with isoamyl acetate: As judged by peak heights, vapor sampled from the outlet port of the saturator was equal to or within 9507o of the concentration of vapor sampled from the head space of a bottle of amyl acetate maintained in the 19 ° C water bath.

Saturation and degassing times of the system were determined by placing the flame ionization detector in series with the outflow of one stimulus channel. Saturation and degassing times varied depending upon the test concentration and air flow rates. With the flows used in this study, saturation and degassing to approximately 95070 of baseline values occurred within 10-15 min. In practice, at least 20 min were allowed for saturation or degassing after any adjustments were made in odor concentration.

A series of control tests were used to evaluate whether extraneous, non-gaseous cues were available for discriminative responding during the detection and discrimination tests. These included testing trained rats with both stimulus channels set for the same concentration of the same odor (one channel was arbitrarily designated as S+), turning the vapor generator off during a test session and testing bilaterally bulbectomized rats. Normal rats trained on an odor-detection task performed at chance levels when deprived of differential odor cues and each of seven bilaterally bulbectomized rats showed no retention and no relearning of a preoperatively learned odor detection task. In addition, Slotnick and Schoonover (67), using a similar apparatus, found that in tests for absolute detection threshold for isoamyl acetate performance accuracy was a function of stimulus concentration. These results demonstrate that only vapor cues were used for discriminative responding.

Surgical Procedures

Rats were anesthetized with sodium pentobarbital (40 mg/kg) supplemented with ether or with 7°7o chloral hydrate (0.35 g/kg) supplemented with ether. The animal's head was clamped into a Kopf stereotaxic machine and the scalp was infiltrated with 0.2 ml of 2070 Xylocaine. In most experimental animals the left olfactory bulb was exposed and aspirated with the aid of a surgical microscope. Special care was taken to remove all olfactory bulb tissue, including the posterior medial aspect of the bulb that extends under the frontal cortex, and to expose and transect all nerves coming through the crib- riform plate. The medial surface of the contralateral olfactory bulb was visualized but was not damaged.

In unilateral bulbectomized rats lesions of the lateral olfactory tract and/or partial transections of cortex were made in the contralateral hemisphere. Several methods were used to make these lesions. In some cases a 3-4 mm wide knife, constructed from a stainless steel razor blade, was lowered under stereotaxic control through the brain to the base of the skull. In other cases the lateral-posterior surface of the olfactory bulb and the anterior lateral aspect of the piriform cortex were exposed and the lateral olfactory tract was identified and transected by gentle aspiration or with a fine surgical knife. Control rats for these surgical procedures had knife cuts that extended through the lateral aspect of the right hemisphere


but did not interrupt the AC or LOT, or had aspiration lesions of frontal cortex dorsal to the rhinal fissure.

In a few cases (as described below) the olfactory bulbs were left intact and bilateral aspiration lesions were made in the olfactory cortex.

Histological Control

At the termination of testing, most rats were deeply anesthetized with sodium pentobarbital and perfused through the heart with saline followed by 1007o formalin. The brains were kept in 30°7o sucrose/10% formalin for 2-3 days and cut at 40 microns on a freezing microtome. If there was extensive damage at the base of the brain, the brain was embedded in gelatin prior to sectioning. Every section or every other section from the olfactory bulb(s) through the lesion area was saved, moun- ted on gelatin coated glass slides, and stained with cresyl violet for cells (most cases) or with Sudan Black B for myelinated fibers.

Some rats, in the later part of the study, were used to assess remaining connections between the olfactory bulb and the forebrain. A total of 0.5/A of 20°7o horseradish peroxidase (HRP) was injected into 2-4 sites in the rostral half of the olfactory bulb. The animals were perfused 24-48 hours later and the tissue was processed according the method of Mesulam (38).

All tissue sections were inspected microscopically and drawn using a microprojector. Sections from HRP studies were examined microscopically with polarized light. The lesioned areas were plotted on a standard set of closely spaced drawings made from the Slotnick and Hersch (63) atlas of the rat olfactory system. The sections were inspected again 3 months later and the lesion sites again plotted. Cases in which there were significant differences between the first and second analyses with regard to the extent or position of the lesion were evaluated again.

Groups

Five rats served as sham-lesioned controls and seven rats were bilaterally bulbectomized. Animals with unilateral bulbectomy plus lesions of the contralateral hemisphere were divided into separate groups based on the histological outcomes as follows: Rats with lesions to non-olfactory cortex (group LC, N = 9); rats with partial transection of the LOT (group Partial-LOT, n = 7); rats in which the LOT was completely transected (group LOT, n = 7); rats with complete transection of the LOT and partial transection of the AC and lateral olfactory tubercle (group LOT/Part ial AC, n = 5); and rats with complete transection of the LOT and AC (group LOT/ AC, n = 5). In addition, there were other rats that were not tested on all tasks and in which the olfactory bulbs were left intact. These included rats with lesions aimed at bilateral aspiration of the anterior piriform cortex (n = 4), transection of the anterior limbs of the anterior commissure (n = 5), stereotaxic lesions of the olfactory tubercle (n = 3) and several mis- cellaneous cases.

Histological A nalyses

For HRP experiments the outcome of interest was whether anterogradely transported reaction product was observed in olfactory cortex caudal to the transection of olfactory pathways. To minimize false negatives, only cases that had good transport of reaction product to projection targets of the main olfactory bulb (layer la of piriform cortex and the plexiform layer of the olfactory tubercle) rostral to the lesioned areas are described. In these experiments the pattern of anterograde

transport in sections rostral to the lesion was essentially identical with that reported in prior studies of bulbofugal efferents (see, e.g., 42) and is not described in the Results section.

Anatomical terminology for the olfactory cortex generally follows that used by Haberly and Price (26,27), Krettek and Price (29,30) and Slotnick and Hersch (61). However, it was seldom possible to describe accurately the postition of lesions in the anterior-posterior or medial-lateral plane with regard to anatomical structures because the damage produced by aspiration lesions or knife cuts did not respect anatomical bound- aries. The anterior piriform cortex, the area in which most lesions occurred, is fairly homogeneous in structure and, more rostral, is continuous with the lateral aspect of the anterior olfactory nucleus. The anterior-posterior position of most lesions were defined with regard to 3 frontal levels; the olfactory peduncle (rostral to the beginning of the olfactory tubercle), the rostral one-third of the olfactory tubercle and the posterior olfactory tubercle. As used in the present study, the olfactory peduncle refers to the region that extends from immediately posterior to the bulb to the junctional area between the pars dorsalis of the anterior olfactory nucleus and the anterior piriform cortex. Frontal levels are also defined with regard to the coordinates of the Slotnick and Hersch atlas of the rat olfactory system.

In Results section, the sham-operated rats and groups with one olfactory bulb removed and lesions in the contralateral hemisphere (groups LC, Partial LOT; LOT, LOT/Part ia l AC, and LOT/AC) are described first. Except where noted, the olfactory bulbectomy in these animals removed the entire olfactory bulb and extended into the rostral AON but did not damage the piriform cortex or olfactory tubercle. Other cases, which do not fit within these categories, are described sepa- rately.

RESULTS

Sham-Operated Rats (Group Sham)

The five sham-lesioned rats had no brain lesions. Each had perfect or near perfect retention of the isoamyl acetate detection problem (mean errors: 3.6) and each reached criterion performance in the first session of the 0.05070 detection test, the intensity difference test and the two-odor discrimination task. A summary of postoperative scores for this and other groups in the study is given in Fig. 3 and the performance of two sham-lesioned rats is shown in Fig. 4.

Lesioned Control Group (Group LC)

Each of the nine rats in this group had the left olfactory bulb removed and had a knife cut or aspiration control lesion in the right hemisphere. In most animals, the removal of the left olfactory bulb was accompanied by damage to the ipsilateral frontal pole cortex and the rostral half of the anterior olfactory nucleus. In two cases, the lesions were more extensive and extended into the rostral half of the olfactory tubercle and, in one rat, the entire olfactory tubercle on the left was destroyed.

In seven of these rats, control lesions were made in the right hemisphere by a 3-ram wide knife lowered in the frontal plane at the level of the anterior olfactory tubercle. The knife was lowered to a point just dorsal to the anterior limb of the anterior commissure (e.g., Fig. 5, RI0 and R14). In the remaining two rats a small segment of cortex dorsal to the rhinal fissure was removed by aspiration (e.g., Fig. 5, R40). Except for the position of the lesion, these surgical procedures


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were identical to those used in a number of experimental rats described below.

This group made slightly, but not significantly, more errors than sham controls in the postoperative tests (Fig. 3). Like animals in the sham group, lesioned controls had excellent retention of the detection task and rapid acquisition of the other three postoperative tests. There was no relationship between the extent of the damage in the right or left hemisphere and behavior.

Rats with Partial Transection of the LOT (Group Partial-LOT)

The seven rats in this groups had the left olfactory bulb removed and a knife-cut lesion in the right hemisphere. The damage produced by the knife was similar to that of lesioned controls except that the knife cut extended to the base of the

brain and transected part of the LOT. Damage to the LOT in the right hemisphere ranged from transection of all but a small medial component of the tract to transection of only the lateral third of the tract. The anterior commissure and olfactory tubercle were not damaged. The frontal level of the lesions were similar to those in group LOT described below.

The performance of these rats was indistinguishable from those of lesioned controls; each showed excellent retention of the detection task and rapid acquisition in the other three tests. Performance variability within this group was not related to the extent of damage to the LOT or frontal level of the lesion.

Rats with Complete Transection of the LOT (Group LOT)

The seven rats in this group had the left olfactory bulb removed and a knife cut or aspiration lesion of the LOT in the right hemisphere. The lesions were similar to those of the partial LOT group except that the LOT on the right was completely transected. The transection was at the caudal aspect of the olfactory peduncle in one rat (Fig. 6, R36) and at the level of the rostral tubercle in the other cases. The anterior commissure on the right was not damaged and, except two cases in which the lesions encroached upon the most lateral aspect of the olfactory tubercle (Fig. 6, R7; Fig. 7, R23), there was no damage to the tubercle. In three cases the lesions were quite discrete and transection of the LOT was produced with minimal damage to surrounding cortex (e.g., Fig. 6, R37; Fig. 7, R39). In the remaining four cases the lesions were larger and transected all or all but the most lateral aspect of the piriform cortex (e.g., Fig. 6, R7; Fig. 7, R.23).

Two rats (R7 and R2) in this group were used in HRP experiments (as described in the Method section). Rostral to the LOT transection, anterogradely transported HRP reaction product filled the superficial aspect of the external plexiform layer of the piriform cortex and olfactory tubercle. Caudal to the transection, reaction product filled the external half of the plexiform layer of the olfactory tubercle but was absent in the plexiform layer of the adjacent piriform cortex. Reaction product was present in the posterior aspect of the tubercle, approximately 1.5-2 mm posterior to the level at which the LOT was transected. Light reaction product was seen in the external plexiform layer dorsolateral to the LOT in one case. In the tubercle, the reaction product extended laterally to the medial edge of the LOT (Fig. 14).

LOT rats made significantly more errors than lesioned controls in the retention test and in the acquisition of the 0.05% detection task (p < 0.01 for each comparison). They made slightly, but not significantly, more errors than controls in acquisition of the intensity difference test and the two-odor test (Fig. 3).

On the retention test most LOT rats performed at or near chance in the first 20-80 trials, but then rapidly reacquired the detection task. Performance, as measured by errors to criterion on the retention test, was not related to the frontal level at which the LOT was transected or to the extent to which the lesion transected the piriform cortex. Scores on the other three tests were not related to performance on the retention test, level, or extent of the lesion.

Rats with Transection of the LOT Plus Damage to the Anterior Commissure (Group LOT-Partial A C)

The five rats in this group had the left olfactory bulb removed and a knife cut or aspiration lesion of the LOT in the right hemisphere. The lesions were similar to those of rats in


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the LOT group except that the damage from the knife cut or aspiration lesion extended medially to interrupt the lateral aspect of the anterior limb of the anterior commissure and the lateral aspect of the olfactory tubercle. In two cases (Fig. 8, R27; Fig. 9, R21) the lesion completely transected the piriform cortex, the lateral 1/3 to 1/2 of the anterior limb of the anterior commissure and the lateral 1/3 of the olfactory tubercle. A third case (Fig. 9, R17) was similar except that the lesion spared the dorsal (sulcal) piriform cortex. In the fourth rat (Fig. 8, R28), the lesion, produced by aspiration, was con- siderably wider in the frontal plane and extended into the AON. There was only slight damage to the lateral aspect of the limb of the anterior commissure but the lateral 1/3 of the AON was removed, as was the rostral 2-3 mm of the piriform cortex. In the fifth rat (Fig. 8, R37A) there was bilateral destruction of the anterior olfactory cortex. Aspiration lesions in the right hemisphere destroyed the lateral aspect of the

AON, invaded the lateral half of the AC and rostral olfactory tubercle and the anterior piriform cortex. In the left hemisphere, damage from the bulbectomy extended posteriorly to destroy almost the entire AON, anterior piriform cortex and medial half of the rostral olfactory tubercle.

One animal in this group (R27) received a bulbar injection of HRP. The results were similar to that seen in rats of the LOT group (Fig. 14). Posterior to the transection, reaction produce was observed through most of the olfactory tubercle but not in the piriform cortex.

Each of these rats had a severe deficit in the detection and two-odor discrimination tasks (Figs. 8, 9). They performed at or near chance for the first 200-1000 postoperative trials before reaching reliable performance of at least 75% correct responding. With additional training each reached criterion performance on the detection task. Performance on the intensity difference and 2-odor discrimination tasks were signifi-


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cantly worse than that of lesioned controls (p < 0.005 in each case, Fig. 3). Three rats did not reach criterion on the intensity difference test and, on the two-odor discrimination task, four showed little or no acquisition in the first two sessions but obtained scores of 80o7o in the third session (after 430 or more trials). The fifth rat, the one with most extensive lesions (Fig. 8, R37A), performed at chance on all 600 trials of the two- odor discrimination problem.

Retention scores within this group were related to the level at which the transection was made and/or the extent to which the AC was damaged. Based on lesion placement and size, three subgroups could be identified. The first consisted of the two rats that had the most rostral lesions (Fig. 8, R37A and R28). In both, the LOT was transected at the level of the most posterior aspect of the olfactory bulb and the lesion destroyed all (R37A) or most (R28) of the lateral half of the AON. In R28 there was also damage to the medial aspect of the olfactory tubercle from the olfactory bulbectomy in the contralateral hemisphere. These rats had the poorest performance in

the group. A second subgroup contains the rat that had the most posterior lesion. The lesion, produced by a knife cut, was very narrow in the frontal plane, invaded only the lateral 1/3 of the AC and extended only into the most lateral aspect of the OT. This rat had the best performance of the group, although it made many more errors than did rats with discrete LOT lesions (Group LOT). The last subgroup, consisting of R27 (Fig. 8) and RI7 (Fig. 9), had lesions that were intermediate in position and extent to those of the first two subgroups. The performance of these two rats was also intermediate to those of rats with the more rostral and more posterior lesions. Within this subgroup, the rat whose lesion invaded the lateral half of the AC (Fig. 8, R27) performed more poorly than did the one with a more rostral lesion but with less damage to the AC (Fig. 9, Rl7). However, performance of these rats in the remaining tests was not related to level or extent of the lesion, although the rat with the most extensive lesion (R37A) was the only one that performed at chance on the two-odor test.

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group. This rat (not included in the control groups) had lesions that were similar to those of the LOT-Partial AC group: The lesion transected the entire piriform cortex and the lateral 3/4 of the AC at the level of the rostral olfactory tubercle. However, in the right hemisphere a segment of the posterior medial part of the olfactory bulb was spared by the bulbectomy procedure. This rat made only 26 errors on the retention test, 0 errors on the 0.05% detection, 190 errors on the intensity difference test and 73 errors on the two-odor test. HRP results for this rat was similar to that for R27: Caudal to the transection, reaction product was evident in the plexiform layer of the lateral and medial olfactory tubercle, but was absent in the plexiform layer of the piriform cortex. Reaction product in the lateral olfactory tubercle extended medially to the medial aspect of the LOT.

Rats with Complete Transection of the LOT and A C (Group LOT-AC)

The five rats in this group had the left olfactory bulb removed and a knife cut or aspiration lesion in the right hemi-

sphere. The lesions were similar to those of rats in group LOT/Partial AC except that they transected both the LOT and AC. As in group LOT/Partial AC, 3 subgroups could be identified. In the first (Fig. 10, R15 and R24), the lesions transected the entire olfactory cortex and the anterior limb of the anterior commissure. Both rats performed at chance in postoperative olfactory tests but were able to perform well when tested on the preoperatively learned visual discrimination. These rats were apparently anosmic. The lesions of the two rats in the second subgroup (Fig. 10, R4; Fig. 11, R25) were more posterior or less extensive. In R4 the lesion was made by lowering a knife through the hemisphere and was similar to that of R15 but was quite narrow in the frontal plane, and did not transect the olfactory tubercle. This rat failed to reach criterion on the retention test in 2000 trials, but it did achieve accuracy scores of 70%o to 80%o on isolated blocks of trials. In R25 the lesion transected almost all of the olfactory cortex but was more posterior and centered at the level of the posterior aspect of the OT. This rat's retention performance was slightly better than that of R4; it did not reach criterion on the retention test but, in the last 1000 trials,

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produce a moderate retention deficit in a simple odor detection task.

Three rats had lesions of the olfactory tubercle (group OT). In one, almost the entire olfactory tubercle was destroyed bilaterally by a series of stereotaxically-placed anodal lesions (Fig. 12, R142). The lesions transected the anterior limb of the anterior commissure, the medial aspect of the LOT and extended into the medial aspect of the piriform cortex in one hemisphere, but produced little or no damage to these structures in the other hemisphere. The other two rats had similar lesions except that the rostral 1/4 of the olfactory tubercle was spared. These rats were tested on retention, the intensity difference problem and the two-odor problem. They performed about as well as controls (Fig. 3).

In four rats an attempt was made to aspirate the greater part of the piriform cortex bilaterally. In one of these (R35) the olfactory peduncle was completely transected bilaterally

at the level of the most posterior aspect of the olfactory bulb. The postoperative performance of this animal in 2000 postoperative trials was identical with that of bilaterally bulbectomized rats.

The remaining three rats (group PC) had extensive lesions of piriform cortex. In each case the LOT was transected bilaterally at the caudal level of the AON and the aspiration lesions destroyed almost the entire anterior piriform cortex, transected the anterior commissure in one hemisphere, the lateral 1/3 of the AC in the other hemisphere and invaded the lateral aspect of the olfactory tubercle in both hemispheres. None of these rats were anosmic but each had a severe deficit in relearning the detection problem and made 321 to 868 errors before reaching reliable performance at or higher than 75°7o correct responding (Fig. 13). In general, the performance of these rats was similar to those in group LOT/Partial AC.

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bulb removed and a transection of the medial 2/3 of the olfactory peduncle in the right hemisphere. The lesion on the right invaded the posterior and medial aspect of the olfactory bulb, transected the AC, the medial aspect of the LOT and invaded the medial 1/3 of the piriform cortex. This left only the remaining LOT and lateral aspect of the piriform cortex intact at the level of the lesion. HRP results demonstrated excellent transport of reaction product to the lateral olfactory peduncle and, caudal to the lesion, the piriform cortex and lateral 1/3 of the olfactory tubercle. The performance of this rat was identical with that of controls; it made only eight errors in the postoperative retention test and 38 errors in learning the two-odor discrimination.

D I S C U S S I O N

Nature and Specificity of the Behavioral Tests

Although control procedures, described in the Method section, established that discrimination performance was not me- diated by non-vapor stimuli, it is important to consider whether trigeminal cues could have been used in these tests. The fact that bilateral bulbectomy resulted in anosmia provides some control for trigeminal cues but, because the eth- moidal branch of the trigeminal nerve, which runs along the lateral surface of the bulb, would be transected when the olfactory bulb is removed, the surgery would partly deprive the nasal vault of trigeminal input. However, it is unlikely that trigeminal cues were available or used because the concentration of the isoamyl acetate vapor stimulus was below the trigeminal threshold, as determined in electrophysiological stud-

ies (56,71,72), and because anosmia was produced by retrobulbar lesions that would not have disrupted trigeminal sensory afferents.

It also seems likely that behavioral deficits in experimental rats were due to changes in olfaction rather than changes in motivation, impaired ability to perform the operant task, or to other non-olfactory effects. By the end of the 3-week postoperative recovery period all animals appeared to be healthy, active, and motivated during the behavior tests. Further, control tests with visual stimuli indicated that deficits in discrimination were specific for odors: For rats that had been trained preoperatively on both visual and odor cues and performed at chance levels on the postoperative odor detection task, rein- troduction of visual cues resulted in a sudden improvement in performance (Fig. 10, open circles). Thus, the lesion-induced deficits reflected changes in some aspect of olfactory function.

The primary measure of olfaction in this study was performance on the isoamyl acetate detection test. This test was designed to be simple and to provide a measure of residual olfactory function in rats with large lesions of the olfactory system. The test required only that the animal detect the presence of a relatively high concentration of an odor. Normal rats typically learn this detection task in a single session and with few errors (58). The 0.5% stimulus concentration used is approximately 4-5 orders of magnitude above the absolute detection threshold for isoamyl acetate (67) and probably the strongest concentration that could be safely employed as a pure odor stimulus; higher concentrations would approach trigeminal threshold for this vapor (56,71). Because of these factors, and because rats received extensive overtraining on


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this detection task, the postoperative detection test probably did not (and was not intended to) provide a very sensitive measure of small lesion-induced deficits in olfaction. Conse- quently, even moderate deficits in this test probably reflect a significant sensory loss.

Olfactory Deficits

Perhaps the two most notable outcomes of the present study were (a) that discrete transection of the LOT or AC, or lesions of the olfactory tubercle resulted in only small deficits in odor detection and (b) that anosmia or very severe deficits in olfaction were obtained only by massive lesions of the olfactory cortex or knife cuts that transected almost all of the olfactory peduncle or cortex.

Rats in which only the LOT was transected at a level caudal to the olfactory peduncle had surprisingly small deficits in olfaction. Their performance was significantly worse than controls but the absolute magnitude of these deficits was small

and several of these rats had scores that were similar to those of controls.

This outcome stands in contrast to several reports that transection of the LOT produces severe deficits in olfaction. Long and Tapp (31), using instrumental conditioning, found that rats with complete transection of the LOT failed to show a preference for the vapor of food odor when hungry and failed to learn an odor-signalled (vapor of wet food mash) avoidance response or maze discrimination. In fact, the scores of rats with LOT lesions were similar to those of olfactory bulbectomized rats. Thompson (70) reported that rats with transection of the LOT plus lesions of the piriform cortex had no retention of a preoperatively learned shock-motivated acetone detection task tested in a choice-box apparatus. The lesions, as illustrated in these two reports, were similar in extent and position to those of a number of animals in the LOT group of the present study. The discrepancy between the present results and those of Long and Tapp, and of Thomp- son, is probably related to the specificity and adequacy of the


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FIG. 11. Performance accuracy in blocks of 20 trials and representation of lesions for the remaining two rats in group LOT/AC. Open circles represent performance in 20-trial blocks of a visual discrimination which rats had learned prior to surgery. The lesions in R16 were made using a stereotaxically directed knife; those in R25 were made by aspiration. In each case the olfactory bulb was removed in the contralateral hemisphere.

tests used. In the Long and Tapp study even partly bulbectomized rats failed to learn these tests and the tests themselves failed to discriminate between rats with discrete LOT lesions and those with complete or nearly complete removal of the olfactory bulbs. The poor performance of experimental animals in the Long and Tapp study may be because weak odor stimuli were used, or because there was inadequate training or incomplete stimulus control of behavior. In the Thompson study, animals were tested only for retention and the test was terminated when the animal made twice as many errors as it did in initial acquisition. Because rats made only approximately 10 errors to learn the discrimination preoperatively, the postoperative test procedure must be viewed as inadequate to meaningfully assess the magnitude of the deficits. In addition, in the Thompson study, rats with a variety of other lesions, not directly related to primary olfactory projections, also had no retention of the task.

Cattarelli (11) has examined the effects of transecting the LOT or AC on multiunit activity of bulbar mitral cells, awak- ening effects of biologically significant odors and responsive- ness of behaving rats to odor stimulation. The results were somewhat complex but there were severe deficits for some measures. Rats with the LOT transected failed to show normal "emotional" reactions to the odor of predators (measured by disruption of runway behavior), had reduced mitral cell unit activity in response to predator and control odors and had poor or no odor discrimination (measured by preference tests). However, at least some odors were effective in awaken- ing experimental rats and it was evident that olfactory function was present in lesioned animals. Based on these and related results on the anterior commissure, Cattarelli (12) made the interesting suggestion that the LOT, which was likened to

a lemniscal system, has an important role in odor discrimination during vigilance while the anterior commissure pathway, which was likened to a reticular sensory system, is particularly important in odor-induced arousal or attentive behavior. While a discussion of Catarelh's evidence for this proposal is beyond the scope of the present paper, at least certain outcomes of that study are not supported by our results. Thus, in the present study there were few differences in odor detection and discrimination between rats with LOT and those with AC lesions and both groups performed nearly as well as controls. As in many prior studies, the behavioral assays used by Cat- tarelli were probably inadequate to demonstrate residual function under optimal tests conditions.

Marked deficits or changes in olfactory guided species- specific behavior have also been found after transection of the LOT. Interruption of the LOT has been reported to produce severe deficits in mating behavior of male hamsters (13,35,36), to disrupt the Bruce effect in female mice (48) and to produce effects similar to that of olfactory bulbectomy on rat maternal behavior (20). In studies in which these LOT transections were illustrated, the lesions appear to be similar to those of rats in the present LOT group (20,35). The species- specific behaviors examined in these studies are known to be strongly dependent upon smell (39) and it is reasonable to assume that the behavioral deficits were specific to an olfactory dysfunction. This assumption is also supported by the finding that disruption of mating can be produced in animals with unilateral posterior LOT transection if the contralateral olfactory bulb is removed (14).

The difference in outcomes between the present study and those that have reported substantial deficits in species-specific behaviors from similar lesions of the LOT probably reflects


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FIG. 12. Performance accuracy in blocks of 20 trials and representation of lesions for one rat in group OT (R142) and one rat in group AC (R63). The olfactory tubercle lesions were made using multiple anodal current coagulations. The anterior commissure lesions were made using a stereotaxically directed knife. The knife extended through and approximately 1 mm below the anterior limbs of the anterior commissure. Both olfactory bulbs were intact in these cases.

the fact that the full consequences of the lesion are not revealed by the discrimination tests we used. Other measures, such as tests for olfactory sensitivity may be needed to assess more adequately the degree to which discrete transection of the LOT disrupts learned olfactory behavior. Of course, it is conceivable that animals with transection of the LOT may have had specific anosmias for odors not tested in this study. However, it seems more likely that the changes in species- specific olfactory guided behaviors resulted from a discon- nection of olfactory input to brain areas involved in the arousal or execution of these acts and not from a specific anosmia (13). Certainly, there are important limitations in using only a learning task or only an olfactory-dependent species-specific behavior to assess the functional role of an olfactory projection.

Lesions that transected multiple pathways from the bulb and peduncle produced much greater deficits in olfaction. Thus, rats in group LOT-Partial AC had no retention of the preoperatively learned detection task and most required hun- dreds of trials to relearn the task. However, these animals were not anosmic; they achieved the 90% criterion level in the detection task and most reached criterion performance or performed at better than chance levels on two-odor discriminations in the allotted number of trials. Their increased error scores were due to relatively slow acquisition rather than an inability to detect or discriminate the odors used in these tests. The more severe deficits in these animals were undoubtedly because the lesions transected a greater number of bulbar efferents than did those in the LOT group. Because AON projections that terminate in the ipsilateral olfactory cortex ex-

tend from the part of the AC that was damaged in these rats, the lesions probably interrupted both direct (LOT) and indirect (AON) projections to the ipsilateral olfactory cortex. However, as would be expected on the basis of the anatomical connections discussed above, and as confirmed by the HRP results in several of these rats, the lesions spared some direct bulbar projections, notably those to the olfactory tubercle and to the TT (Fig. 14). In addition, but not confirmed by the present HRP study, these animals (and those in group LOT) may have had olfactory input to the contralateral hemisphere via the AC. The present experiments do not allow the contri- butions of these remaining projections to be identified, but several lines of evidence indicate that contralateral projections were not necessary for olfactory function in these rats. Thus, in preliminary studies (Slotnick and Risser, unpublished observations) we found that transection of the commissural part of the AC had little or no effect on olfactory performance of unilaterally bulbectomized rats in which the contralateral LOT had been transected. Also, it is unlikely that these contralateral projections existed in R37A (Fig. 8) because the targets for these projections (the anterior olfactory cortex and AON) were destroyed in both hemispheres. The performance of this rat was comparable to that of several other rats in group LOT Partial AC in which the olfactory cortex on the bulbectomized side was intact. It is thus likely that the performance in rats of group LOT Partial AC was supported largely by direct projections from the olfactory bulb to the ipsilateral olfactory tubercle, projections to olfactory cortex from the AON and/ or projections to olfactory cortex via the TT.

Among experimental rats, only the two in the LOT-AC

O L F A C T O R Y P A T H W A Y S A N D T H E SENSE OF SMELL 469

100

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FIG. 13. Performance accuracy in blocks of 20 trials and representation of lesions for the three rats in group PC. The lesions were made bilaterally but only the hemisphere with the smaller lesion or less extensive damage to the olfactory cortex is illustrated. In each case the lesion in the contralateral hemisphere was somewhat larger and transected all or most of the AC and invaded the lateral half of the OT. These lesions were made by aspiration. Both olfactory bulbs were intact in these cases. *Indicates a test that was not given.

group in which lesions transected the entire olfactory cortex appeared to be anosmic (i.e., their performance was similar to that o f bulbectomized rats). Interestingly, these transections probably spared bulbar projections to the TT and any connections that it may have with the anterior continuation of the hippocampus and rostral olfactory tubercle (1). This suggests that these inputs alone are unable to sustain detectable olfactory function. Other rats, in which both the LOT and AC were transected, performed largely at chance in 2000 postoperative retraining trials but, because they obtained scores o f 70°7o or higher on isolated blocks o f 20 trials, some residual olfactory function was probably present. Because the transections spared only a small medial segment o f the A O N or olfactory tubercle, it appears that some olfactory function may be medi- ated by very few connections to olfactory cortex. These outcomes are not in agreement with those o f Cattarelli (I 1) that rats with discrete lesions of the LOT and AC did not respond

to odors in a behavioral task. The lesions illustrated in Cattar- elli's study would have spared direct bulbar input to the olfactory tubercle and rats with even more severe lesions in this study were able to detect isoamyl acetate vapor. Again, it is likely that the behavioral tests for odor function used by Cattarelli were insensitive to residual olfactory function revealed in our tests. The present results are in accord with the finding of McBride, Slotnick, Graham, and Graziadei (37) that rats with destruction of approximately 90% of the olfactory bulbs were able to detect odors.

Nature of the Behavioral Deficit While the simplest explanation for deficits in odor detec-

tion and discrimination obtained in the present study is that interruption of olfactory pathways decreased sensory capacity, several observations suggest that this explanation is insuf- ficient. Within different groups there was no relationship be-

470 SLOTNICK A N D S C H O O N O V E R

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FIG. 14. Photomicrographs of the lesion in R7 from group LOT. The LOT lesion was made by aspiration and HRP was injected into the ipsilateral olfactory bulb 24 h prior to sacrifice. The sections were reacted according to the procedures of Mesulam and lightly counterstained with thionin. A-D, light microscopic photomicrographs showing (A) the most rostral extent of the lesion, (B) the largest extent of the lesion, (C) the last section in which the LOT was completely transected and (D) a section, approximately 0.3 mm posterior to C, in which the LOT is intact. The lesioned area on the superficial aspect of the brain is filled with the gelatin embedding material. Anterograde transport of HRP reaction product is evident in the plexiform layer of the olfactory tubercle (B-D) but not in the piriform cortex. (E) higher magnification polarized light photomicrograph of the lateral olfactory tubercle and LOT area in D. Note the dense anterograde reaction product in the olfactory tubercle (solid arrows) and its absence in the plexiform cortex deep to the LOT. The anterograde reaction material terminates abruptly at the junction of the olfactory tubercle and piriform cortex (open arrow)• Light spots in the LOT are artifacts due to scattering light by the myelinated fibers; no evidence of anterograde HRP transport was found in the LOT or piriform cortex at this level.

tween per fo rmance on the first pos topera t ive test and on subsequent tests. In fact, the pe r fo rmance of mos t exper imental rats improved over the test series despite the fact tha t the tests were graded in difficulty. This was part icular ly clear in the LOT-Par t ia l AC group. All rats in this group per formed much bet ter on the 0.05°7o detect ion task than in the initial 0.5070 re tent ion test (Fig. 3). Fur ther , the pe r fo rmance of most was bet ter in the two-odor d iscr iminat ion task t han in the initial trials of the re tent ion test. Improvemen t was also evident in rats in the LOT group. They made approximate ly six times as many errors as did controls on the odor detect ion tests but per formed as well as controls on the subsequent two- odor discr iminat ions. Thus, the deficits in the first postoperative test do not simply reflect a reduct ion in sensory capacity. Observat ion of the animals dur ing postoperat ive tests suggests

tha t , in initial sessions, pe r fo rmance accuracy was related to at tent iveness to the st imulus. In the first pos topera t ive trials we observed tha t animals in the LOT and LOT-Par t ia l A C groups tha t pe r fo rmed poorly did not adequately sample the st imulus before responding. Frequently, responses were made dur ing the final valve period (prior to the in t roduc t ion of the st imulus) or immediately af ter the end of the f inal valve per iod but , apparent ly , pr ior to sampling the st imulus. For rats in the LOT group, improvement over trials was clearly correlated with more careful s t imulus sampling. This was also t rue of rats in the LOT-Par t ia l AC group a l though, in these rats, errors also occurred on trials in which the an imal appeared to sample carefully before responding. We also no ted tha t several olfactory bulbectomized rats sampled vigorously on mos t trials (74). Thus, inadequate sampling of the st imulus may

O L F A C T O R Y P A T H W A Y S A N D T H E SENSE OF SMELL 471

account for initial errors in the retention test but the presence of sampling behavior alone is not predictive of performance accuracy. Nevertheless, in future studies it might be useful to obtain a quantitative measure of odor sampling. As shown recently by Youngentob, Mozell, Sheehe, and Hornung (75) and in preliminary studies in this laboratory (60), odor sampling, particularly the difference in sample duration on S + and S- trials, is a sensitive measure o f detection and discrimination behavior and may provide additional informat ion concerning olfactory deficits in experimental animals.

These observations suggest that in addition to a change in sensory capacity, other factors such as decreased attentiveness may contribute importantly to the greater error scores of lesioned rats. Such "non-sensory deficits" might reflect a lesion- induced reduction in the salience o f the stimulus (40,58,62,65). It is not known which forebrain areas are important in mediat- ing the salience of odor cues but most areas of the olfactory bulb and even individual mitral cells project over wide areas of olfactory cortex (41,33) and, thus, it is likely that almost any interruption o f olfactory bulb efferents would reduce input into such target structures.

A notable result of the present study and that o f the Mc- Bride et al. (37) report on partial olfactory bulb lesions is the extent to which olfactory function is spared after lesions o f different parts of the system. Certainly the simplicity o f the task, methods of achieving stimulus control and the use of somewhat high concentrations o f odors contribute to these savings. However , savings also may be related to the unique organization of olfactory projections to olfactory cortex. An-

atomical and electrophysiological studies have indicated that the olfactory bulb projections to the olfactory cortex are not topographically organized in a point-to-point fashion but, instead, are broad and overlapping (33). Even axons o f individual mitral cells arborize widely in the A O N and olfactory cortex (41). The manner in which odors are coded at the level of the cortex is not known, but the anatomical organization suggests that information may be represented as spatially dis- tributed patterns [e.g., (21,22,28)].

Haberly (25) has suggested that olfactory information is stored as an ensemble code in the cortex such that any one odor would activate many thousands of cells and each cell might participate in the coding of many different odors. The behavioral savings demonstrated in the present study may reflect the redundancy inherent in an ensemble coding mechanism. These experiments do not provide a direct test of this hypothesis but the findings that discrete lesions of the LOT or AC, or destruction of the olfactory tubercle, produce only moderate and approximately equivalent deficits are consistent with a mass action principle of olfactory cortical function or, as described by Haberly, a mechanism in which widespread areas of the olfactory cortex participate in odor detection and discrimination.

ACKNOWLEDGEMENTS

The authors are pleased to express their appreciation to Paul D. MacLean for his valuable suggestions and support during the course of these experiments and to John Scott and Joseph Price for their many useful comments on the manuscript.

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Olfactory pathways and the sense of smell