Neuroethology of olfactory preference development

Neuroethology of Olfactory Preference Development

Michael Leon

Department of Psychobiology, University of California, Irvine, California 9271 7

SUMMARY

Young mammals come to approach the odor of their mother, a response that facilitates their survival during early life. Young rats induce a cascade of events in their mother to induce the emission of her odor. The pups increase circulating prolactin levels, which increases food intake and the emission of large quantities of cecotrophe containing the maternal odor. This odor is synthe- sized by the action of cecal microorganisms and changes with maternal diet. The diet-dependence of the odor requires the pups to acquire their attraction to the odor postnatally. The acquisition of this preference occurs when an odor is paired with the tactile stimulation that

pups receive during maternal care. The action of the tactile stimulation appears to be mediated by noradrenaline. The development of this type of olfactory attraction is accompanied by changes in the regions of the olfactory bulb that are responsive to the attractive odor. Meta- bolic, anatomical, and neurophysiological changes in response to the attractive odor emerge in such regions of the bulb after early olfactory preference training. 0 1992

Keywords: 14C 2-deoxyglucose, cecotrophe, brain development, dopamine, food intake, mitral cells, morphome- try, noradrenaline, olfaction, olfactory bulb, prolactin.

John Wiley & Sons, Inc.

INTRODUCTION

The growth and survival of young mammals is criti- cally dependent upon the care they receive from their mother. Parental care, in turn, depends upon a close synchrony between mother and young as infants progress rapidly through increasing levels of competence until the time of weaning. In many mammals that have been studied, maternal olfactory cues play an important role in maintaining interactions during the different phases of the parental cycle. The complex nature of mechanisms involved in the production of a maternal odor in Norway rats, as well as the mechanisms for the development of a preference to such odors, has been a major focus of our laboratory. In the coming pages, I will describe the work we have done on this project over the last 20 years. Specifically, I will describe: ( 1 ) the physiological basis for maternal ol-

Received August 12, 1992; accepted August 12, 1992 Journal of Neurobiology, Vol. 23, No. 10, pp. 1557-1573 (1992) 0 1992 John Wiley & Sons, Inc. CCC 0022-3034/92/101557-17

factory attractant; ( 2 ) the experiential basis for olfactory preference acquisition; and ( 3 ) the neurobiology of this type of early learning.

To tell the entire research story that has emerged from my laboratory within the confines of this arti- cle, I restricted the discussion largely to our own data. In doing so, I must apologize in advance for the omission of the many elegant studies done by other laboratories on this and related topics. Be- cause there exist several reviews that cover the breadth of the area (Leon, 1983; Alberts, 1987; Blass and Kehoe, 1987; Hall, 1987; Hofer, 1987; Leon, Coopersmith, Lee, Sullivan, Wilson, and Woo, 1987; Spear and Molina, 1987; Smotherman and Robinson, 1987; Coopersmith and Leon, 1988a; Porter, Balogh, and Makin, 1988), I ask the reader’s indulgence for taking the opportunity to describe this one story in depth.

MATERNAL OLFACTORY ATTRACTANT

Maternal Olfactory Attraction

Altricial mammalian young must either maintain contact with their mother or remain in the mater-

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nal nest in her absence. Norway rat young remain in the nest during their mother’s periodic absences, rapidly orient toward her when she is there, and approach her when she is nearby. Nyakas and En- droczi ( 1970) showed that this ability is at least in part mediated by an olfactory attractant emitted by the mother. Postnatal day (PND) 10 pups approached their mother in a U-shaped maze in preference to either a nonlactating female or a male. Pups also preferred the arm of the maze the mother had previously occupied compared to one that had been occupied by a nonlactating female rat. Pups did not approach the mother’s urine, a warmed arm of the maze, or the mother herself if the pup’s nasal mucosa was anesthetized.

In a parallel study, Leon and Moltz ( 197 1 ) found that almost all the PND16 pups tested in an olfactory discrimination apparatus preferred the odor of their dam to that of a nonlactating female. Pups were able to indicate their preference by crawling across an open field and then descending a small cliff The odor of their own mother and another mother were approached equally in this apparatus. The olfactory nature of the maternal stimulus was demonstrated by the loss of maternal preference when pups were upwind of the lactating females.

Synchrony of Odor Attraction and Emission

In this testing situation, neither PND 1 nor PND 10 pups approached the odor of the mother, even when they could register their preference with a locomotor response that was well within their ability (Leon and Moltz, 1972). It is not clear, however, that they could not register a choice at these ages if a more subtle response was measured. Be- tween PND 12-14, though, pups began to approach the maternal odor. This attraction re- mained intact through PND 27 and waned by PND 4 1. This developmental pattern of attraction held even when the odor of the dam was held relatively constant by allowing pups to choose between a colony dam, 16 days postpartum, and a nonlactating female at each age. Similarly, when stan- dard-age pups (PND 16) were allowed to approach mothers of different lactational ages mothers attracted these pups in the same temporal pattern as with normally aging pups. Mother and young therefore have a remarkable symmetry in the time that dams emit the attractant and the period during which the young will approach it. It also is interesting that both maternal odor emission and the at-

traction by pups persist through the fourth week postpartum because the young must continue to reunite with their dam for nursing bouts until weaning occurs at that time.

Source of the Attractive Odor

The source of the attractant is the mother’s anal excreta (Leon, 1974). Pups preferred the odor of the maternal anal excreta to that of nonlactating females but had no preference for other maternal excretions. In addition, the odor of the anal excreta has a similar temporal pattern of attractiveness as the mother herself over the course of lactation. There is a quantitative and qualitative change in the mother’s anal excreta at the time that it becomes attractive to pups. Specifically, both the amount defecated and the volatile components increase dramatically during the period of maternal attractiveness. The reason for the changes in these parameters is that there is a shift in the proportion of the two kinds of anal excreta defecated by mother rats. The first kind is defecated in hard, dry pellets that are not particularly attractive to the young. The second is a semisolid substance called cecotrophe that is normally completely reingested by nonlactating rats ( Harder, 1949). This material is highly attractive to the young. The reason for both the physical changes in the mother’s excreta, as well as for the increase in its attractiveness, is that mothers greatly increase the amount of unreingested cecotrophe that they emit.

Cecotrophe is produced in the cecum, a large structure pouching out at the junction of the small and large intestine, which becomes greatly enlarged during lactation (Fell, Smith, and Camp- bell, 1963). A portion of the food that passes through this structure is acted on by resident mi- crobes, which facilitates diet utilization and vitamin production upon reingestion (Harder, 1949; Hoetzel and Barnes, 1966). The cecum is also the site of maternal odor synthesis; material taken from the cecum but not above that level in the alimentary canal was attractive to pups (Leon, 1974). Indeed, the material taken from the ceca of both nonlactating females and males was also attractive to pups. If one collects the small amount of unreingested cecotrophe defecated by these rats, it too is attractive to pups. These data suggest that all adult rats produce cecotrophe but lactating females begin to emit sufficient quantities of cecotrophe to get into the environment and attract pups.

Olfactory Pre/erenc'e Development 1559

Mechanism for Odor Emission One reason for the increase in cecotrophe defecation is that mothers greatly increase their food consumption during lactation (Ota and Yokoyama, 1967a, 1967b; Fleming, 1976). When maternal food intake was restricted, cecotrophe defecation and maternal attractiveness decreased, even while lactation was maintained (Leon, 1975). These data support the notion that lactation promotes increased food intake, which in turn promotes the increased defecation of cecotrophe, which carries the attractive maternal odor to the external environment.

Pup Stimuli Control Maternal Odor Emission Maternal food intake is sensitive to litter size and age (Ota and Yokoyama, 1967a, 1967b) and the emission of the maternal attractive odor is similarly affected by litter stimulation. On PND 1, mothers eat relatively small quantities of food, defecate little or no excess cecotrophe, and do not attract pups from afar. By PND 16, however, mothers eat two to three times that of nonlactating rats and greatly increase their cecotrophe emission. These mothers are highly attractive to pups. When mothers are permitted to nurse only PND 1 pups until they are 16 days postpartum, they eat relatively little through day 16, emit no cecotrophe, and do not attract pups. Control dams receiving a new foster litter of advancing age each day ate, defecated, and attracted pups in a manner similar to dams caring for their own normal litters (Moltz and Leon, 1973; Leon, unpublished observations). It was not possible, however, to advance the onset of maternal odor emission by allowing mothers to nurse PND 10 pups starting on day 1 postpartum, but the introduction of weanlings stopped odor emission by mother rats (Holinka and Carlson, 1976), presumably by stopping lactation, thereby decreasing maternal food intake (Ota and Yokoyama, 1967b). Continuing to maintain mothers with PND 14-21 pups maintains lactation (Selye and McKeown, 1934) and high levels of food intake (Tomogane, Ota, and Yokoyama, 1976) and allows mothers to continue emission of their attractive odor (Holinka and Carlson, 1976; Moltz, Leidahl, and Rowland, 1974).

Hormonal Control of Maternal Odor Emission Because maternal food intake and odor production is affected by pup stimulus characteristics, and be-

cause such stimulation increases maternal hor- mone release, we considered the possibility that hormonal action is involved in odor emission. To evaluate this possibility, we systematically elimi- nated hormones known to increase during lactation. Because reduction of some hormones interferes with lactation, experimental mothers in each study had their pups switched with control mothers each day both to maintain the health of pups in the face of diminished lactation and to keep all mothers with equivalent pup stimulation. Neither postpartum adrenalectomy, ovariectomy, nor the combined operation affected maternal food intake, defecation, or odor emission (Leon and Moltz, 1973). However, pharmacological block of prolactin stopped lactation and prevented the increase in maternal food intake, maternal cecotrophe defecation, and maternal attractiveness (Leon and Moltz, 1973; Leon, 1974). Exogenous prolactin, given to mothers whose own prolactin was pharmacologically blocked, restored lactation, maternal food intake, cecotrophe defecation, and maternal attractiveness. When nonlactating female rats were injected each day with prolactin for 16 days, they had increased food intake and increased defecation and became attractive to PND 16 pups (Leon, 1974). These data are in particular interesting given the importance of appropriate pup stimulation both in evoking high levels of circulating prolactin in their mothers and maintaining maternal odors (Moltz, Levin, and Leon, 1969; Grosvenor, Maiweg, and Mena, 1970; Leon and Moltz, 1973).

Microbial Control of Odor Synthesis

In any analysis of natural attractants, it is important to discriminate between odor emission and odor synthesis. Because odor synthesis appears to be present in the ceca of both lactating and nonlactating rats, and the cecum is the site of microbial action ( Raibaud, Dickenson, Sacquat, Charlier, and Moquot, 1966), we first determined whether maternal odor synthesis is under bacterial control. When mothers were allowed to ingest either of two antibiotics, their no longer attracted pups (Leon, 1974) although they continued to defecate as much as controls. Moreover, material taken directly from maternal ceca was not attractive to pups after antibiotic treatment. These data suggest that odor synthesis, but not emission, was blocked by antibiotics.

While the antibiotic action suggested a role for microbial synthesis of the maternal odor, it was

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also possible that these drugs reacted chemically with the odor or suppressed a gland secreting the odor. Therefore, we blocked microbial function without using antibiotics by depriving the cecal mi- crobes of their growth medium. Because cecal bacteria require carbohydrates for their survival, we fed dams a diet in which the only constituent car- bohydrate was sucrose, which is absorbed above the level of the cecum ( Roscoe, 1927; Hopkins and Leader, 1946). This diet also contained the vita- mins normally produced by cecal microorganisms. Mothers on this diet lactated normally but their anal excreta did not attract pups (Leon, 1974). The material taken directly from the ceca of the sucrose diet mothers also did not attract pups, suggesting that odor synthesis, rather than emission, was blocked by this procedure. When complex carbohydrates were restored to the diet, these mothers produced an odor that attracted pups.

Dietary Control of Odor Synthesis

Because the odor is dependent upon cecal mi- crobes and different diets allow different popula- tions of bacteria to prosper (Porter and Rettger, 1940; Smith, 1965), it seemed possible that different diets would produce different maternal odors. The existence of diet-dependent odors could be observed only if olfactory preferences were specific to the odors of dams eating a specific diet. We found that mothers fed different diets produced different odors and that pups were attracted to only the odors with which they were raised (Leon, 1975). Pups raised without a dominant maternal odor (mothers were given the sucrose-based diet that suppressed cecal odor) but given 3 h daily exposure to maternal odor came to prefer that odor. Clearly, mothers produce different odors when they are eating different diets and the attraction to maternal odors is acquired postnatally.

DEVELOPMENT OF AN ATTRACTION TO ODORS BY THE YOUNG

Preference Development to Nonmaternal Odors

If pups develop a postnatal preference for a diet-dependent odor, then they might also come to prefer nonmaternal odors with postnatal experience.

Pups were therefore given prolonged exposure to peppermint odor ( 3 h / day) for the first 19 days of life and then tested for an olfactory preference on day 20. Such experience produced both a preference for that odor and a bias for ingesting food with that odor (Leon, Galef, and Behse, 1977).

Development of an odor preference could be fa- cilitated by exposing pups to the odor briefly with concurrent tactile stimulation designed to mimic maternal contact. This stimulation was provided by brushing pups vigorously either on the perineal region or over the entire body. At this point, we began to use a simpler, but effective test for olfactory preference by observing the time spent over each of two grid-covered, scented trays. Brief (10 min/day j odor exposure on PND 1- 18 coupled with tactile stimulation produced a clear olfactory preference on PND 19 (Coopersmith and Leon, 1984).

Further study demonstrated the associative nature of the learned response (Sullivan and Leon, 1986). Pups were trained with either concurrent odor and tactile stimulation, tactile stimulation alone, odor alone, or left experimentally naive during the 18 days preceding the behavioral preference test. Only those pups with concurrent odor and tactile stimulation developed a preference for the trained odor; odor-only pups were no different than the naive pups. It was possible, however, that pups receiving both tactile and odor stimuli developed the conditioned response not due to an associative process but rather to an increase in arousal or attention produced by the tactile stimulation. To address this issue, we presented a control group with both tactile and odor stimuli, but presented sequentially rather than concurrently (Sullivan, Wilson, and Leon, 1989b). Both groups would therefore receive exactly the same stimulation, but only one group would have the opportunity to form an association between them. In fact, only those pups that had concurrent stimulus presentation, and therefore the opportunity to form an association, learned the olfactory preference. The odor / tactile pairings therefore constitute a classic conditioning paradigm for early learning.

The question of the duration of odor preferences formed early in life was addressed by Fillion and Blass ( 1986). They showed that olfactory preferences learned early in life will affect the sexual behavior of adult, male rats. These data suggest that the early olfactory preferences are long lived, although they may emerge only under special cir- cumstances.

O(factory Preference Development 1561

There appears to be a sensitive period for the development of odor preferences within this learning paradigm. Pups trained with odor/tactile pairings after the first postnatal week do not have a preference for the trained odor on PND 19 (Woo and Leon, 1987 ) . Pups trained on PND 1-4 also do not demonstrate such a preference at PND 19, suggesting that the PND 19 memory requires more training for odor preferences to be preserved until that time. Those pups trained through PND 1-8 and tested on PND 19, however, have a strong preference for the odor.

There also is specificity in this type of early conditioning; training with one odor (peppermint) does not increase the preference for another odor ( cyclohexanone; Coopersmith, Henderson, and Leon, 1986). This type of learning is not restricted to peppermint odor because both cyclohexanone and orange odor were preferred after similar odor/ tactile pairings (Coopersmith et al., 1986; Wilson, Sullivan, and Leon, 1987; Wilson and Leon, 1988a).

Waning of the Olfactory Preference in Rats

To study the termination of pup responsiveness to maternal odor, we first determined more carefully the time course of the waning attraction. Attrac- tion to a standardized maternal odor was strong through PND 24 but began to decline sharply on PND 25 and declined further to a virtual random choice by PND 27 (Leon and Behse, 1977 ) . This is the time of weaning, and young begin producing their own cecotrophe at this time. Because weanlings are eating the same diet as their mother, and because young develop the same enteric bacteria of their dam (Smith, 1965), it seemed possible that weanling rats would begin to produce a cecal odor similar to that of their mother. Indeed, the cecal odor of these weanlings was attractive to other pups. The cessation of the preference for the mother’s odor might be due to their new ability to produce the same odor. Weanling rats deprived of their cecal odor either by surgical removal of their cecum or by their consumption of the sucrose- based diet continued through PND 35 to approach the maternal odor to which they had developed an attraction (Leon and Behse, 1977). These data suggest that rather than losing interest in the maternal odor young may switch their allegiance from their mother’s to their own similar odor.

NEURAL CHANGES INDUCED BY EARLY OLFACTORY LEARNING

Enhanced Neural Response at the First Synapse in the Olfactory System

If pups learn to prefer odors postnatally, there should be a trace of this type of learning in the brain. We decided to look at the first synapse in the olfactory system for such evidence.

This synapse exists in the olfactory bulb glomeruli, large, ball-like structures composed of neuropil (Fig. 1 ). Here, the axons of many olfactory receptor neurons, which transduce the chemical cues into neural signals, synapse with the dendrites of relatively few second-order cells. There are three types of these second-order neurons. The major output neurons of the bulb are the mitral cells. The tufted cells have intrabulbar, and interbulbar, connections, as well as having some connections to the olfactory cortex (Macrides, Schoenfeld, Mar- chand, and Clancy, 1985). The third class of these neurons are the periglomerular cells, which appear to mediate interglomerular inhibition.

For the initial level of analysis, we decided to use a technique that would enable us to observe functional changes throughout the olfactory bulb. The 2-deoxyglucose (2-DG) method served this pur- pose. 2-DG is a glucose analog that is utilized by active cells but incompletely metabolized, accu- mulating in the cells, thereby allowing autoradio- graphic study of its uptake when it is radioactively labeled. The technique allows one to localize and quantify differential responses in the brain.

We used 14C-labeled 2-DG autoradiography to measure relative cellular activity in the olfactory bulbs of 19-day-old rat pups during exposure to the trained odor. One group of pups previously had received concurrent peppermint odor and tactile stimulation for 10 min/day on PND 1-18 and a second group was exposed to clean air with tactile stimulation. On day 19, peppermint-trained pups had 64% higher uptake of 2-DG in response to peppermint in glomerular-layer foci than control pups. The foci are found lateral and 1.5-2.2 mm from the rostra1 pole of the bulb (Coopersmith and Leon, 1984).

The enhanced response is a consequence of associative learning. Only those pups receiving concurrent odor and tactile stimulation had a subse- quent increase in 2-DG uptake in the 2-DG foci (Sullivan and Leon, 1986; Sullivan et al., 1989b; Fig. 2). Neither odor alone or even odor and tactile stimulation given sequentially induced the en-

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olfactory receptor neurons

PERIGLOMERULAR TUFTED CELL

MITRAL CELL

- 11 GRANULE CELL

ExternGI Plexiform Layer

Figure 1 Simplified drawing of olfactory bulb circuitry. Olfactory receptor neurons transduce chemical cues. They then enter the olfactory bulb, where they synapse with second-order neurons in ball-shaped glomeruli. The mitral cells are the dominant output neuron ofthe bulb, although some tufted cells have extrabulbar as well as intrabulbar connections. The periglomerular cells and the granule cells are inhibitory interneurons.

hanced metabolic response. The neural responses to the learned odor match the behavioral responses to such cues.

The enhanced response is odor and site specific. Pups trained with peppermint have an enhanced glomerular response to peppermint, but not to cyclohexanone, which is processed in a different part of the bulb (Coopersmith et al., 1986). Thus, it appears that olfactory preference training induces a response specific to that odor rather than a gen- eral activation of the olfactory bulb. There is also an enhanced response to cyclohexanone in a different part of the bulb if pups have been trained with that odor (Coopersmith et al., 1986). These data parallel the behavioral findings showing the same type of odor specificity after early olfactory conditioning.

Pups raised by mothers have an enhanced 2-DG uptake in response to that maternal odor (Sullivan, Wilson, Wong, Correa, and Leon, 1990). Again, the pattern of neural responsiveness parallels the behavioral responsiveness. Pups will also have an enhanced uptake of 2-DG in response to pepper-

mint odor after that odor had been associated with the mother during PND 1-18 (Sullivan, Wilson, Wong, Correa, and Leon, 1990).

The enhanced response is long lived. Pups given peppermint preference training on PND 1 - 1 8 and tested for 2-DG uptake when reexposed to peppermint on day 90 also show the enhanced neural response (Coopersmith and Leon, 1986). This continued enhanced neural responsiveness may play a role in the long-lived behavioral responsiveness seen in rats (Fillion and Blass, 1986).

Not only is the development of the neurobehavioral response restricted to young rats, it appears to be a sensitive period for its development during the first postnatal week. While the enhanced response is subsequently found after training during the first postnatal week, neither training on PND 1-4 nor training after the first week produces the enhanced response. Odor-stroking pairing in adulthood does not induce the enhanced neural response (Woo and Leon, 1987 ). Again, there is a striking parallel with the behavioral findings of a sensitive period for behavioral preference development.

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Figure 2 ( A ) Time over the trained odor (peppermint) in a two-choice odor preference test by PND 19 rats trained with peppermint + tactile stimulation, odor alone, tactile stimulation alone, or left experimentally naive. ( B ) Relative 2-DG uptake in focal glomeruli of PND 19 pups given identical training regimens.

MECHANISMS UNDERLYING THE ENHANCED NEURAL RESPONSE

Role of Stimulus Availability

The enhanced neural response is not due to increased stimulus availability during testing. Nei- ther the total number of respirations nor the fre- quency pattern of respirations differed between odor-trained and control pups during the 2-DG test on day 19 (Coopersmith and Leon, 1984; Coo- persmith et al., 1986; Sullivan and Leon, 1986; Woo and Leon, 1987). Moreover, when pups had an identical number of equal-volume respirations imposed on their olfactory receptors during exposure to the trained odor only trained pups had an enhanced 2-DG response (Sullivan, Wilson, Kim, and Leon, 1988a).

While the expression of the enhanced neural response is not due to differential respiration, it seemed possible that the acquisition of the neurobehavioral response was due to increased respiration during conditioning. The reinforcing tactile stimulation does increase respiration ( Alberts and May, 1984; Sullivan et al., 1988b), but it was not clear whether this increase was critical for the development of the neurobehavioral response to the conditioned odor. We therefore paired the odor

with high humidity, which does not evoke an increase in respiration, and tested for a behavioral preference and an enhanced glomerular response to the odor on day 19. We found that pups acquired a preference and an enhanced glomerular response to the conditioned odor without an increase in respiration during training (Sullivan et al., 1988b). Therefore, differential respiration of the odor during early olfactory preference conditioning is not critical for development of the altered neurobehavioral response (Do, Sullivan, and Leon, 1988).

Morphological Changes

If the enhanced neural response was not due to increased respiration, perhaps there were intrinsic structural changes associated with the 2-DG foci that underlie this differential response. We therefore looked more closely at the glomerular areas associated with the enhanced 2-DG uptake in odor-trained pups and compared the glomerular morphology to that of control pups.

Alternate olfactory bulb sections of odor- trained and control pups were developed for autoradiography or reacted for succinic dehydrogenase to reveal individual glomeruli. We then aligned the stained sections with the autoradiographs to determine whether there were identifiable structural modifications associated with early olfactory learning. Indeed, there were: The glomerular areas associated with the 2-DG uptake in odor-trained pups had enlarged glomeruli that often protruded into the external plexiform layer (Woo, Coopersmith, and Leon, 1987; Fig. 3 ) .

While there was no difference in the number of glomeruli associated with the focal 2-DG uptake sites, there was a 26% increase in glomerular layer width in the focal areas of 2-DG uptake of odor- trained pups relative to that of control pups (Fig. 3 ) . The glomerular layer outside the 2-DG foci did not differ between groups. There was also a 20% increase in the area of individual glomeruli in the focal 2-DG areas of odor-trained compared to control pups (Woo et al., 1987).

The apparent reason for this increase in the glomerular neuropil is that the early learning increases the number of glomerular-layer neurons associated with the 2-DG foci (Woo and Leon, 199 1 ). Condi- tioned pups had 19% more neurons in the 2-DG foci than control pups. There was no change in the size of the neurons. There was also no change in the density of glomerular-layer neurons because the width of the glomerular layer also increased.

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Figure 3 Width of the glomerular layer associated with the 2-DG high-uptake foci or adjacent regions in pups given early olfactory preference training (peppermint fa- miliar) or control pups (peppermint unfamiliar).

Although the glomerular-layer neuronal population consists of both periglomerular cells and tufted cells, this large increase in neural number was due either completely or in large part to an increase in periglomerular neurons. While there are several possible mechanisms that could underlie this increase in cell number, a particularly inter-

esting one is that early learning saves cells from dying. Indeed, there is a postnatal die-off in the periglomerular population (Frazier and Brunjes, 1988) and the increased neural activity associated with early learning may increase the probability that cells in the 2-DG foci will live.

Mitral Cell Activity

Because there is an increase in the number of pen- glomerular neurons in the 2-DG foci of conditioned pups, and because these neurons are inhibitory interneurons, it seemed likely that the output neurons of the bulb that emanate from these affected glomerular regions would have a suppressed response to the conditioned odor. We therefore re- corded single-unit activity from the portion of the mitral cell layer associated with the active glomerular sites in odor-trained and control pups with the trained odor on day 19 (Wilson, Sullivan, and Leon, 1985; Wilson et al., 1987; Wilson and Leon, 1988a).

There were no differences between groups in the number of responses or the magnitude of the responses to the trained odor by identified mitral cells. The cells of peppermint-trained pups, however, had significantly more inhibitory responses and significantly fewer excitatory responses to peppermint than controls. The response is specific to trained pups, the learned odor, and mitral cells in the region of the peppermint 2-DG foci (Wilson and Leon, 1988a). In addition, the differential re-

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Figure 4 Firing rate of mitral cells either associated with the 2-DG hot spot or distant from the hot spot of trained pups. Trained odor is presented starting at arrow. Cells not associated with the 2-DG foci have a net excitation within 500 ms of the odor presentation, whereas cells associated with the hot spots have a suppression in their firing by that time.

Olfactory Preference Development 1565

sponse of the two groups occurred within 500 ms of the onset of the trained odor (Fig. 4). Because these animals were respiring at about 2 Hz, these data suggest that the differential response is occur- ring within the first respiration of the learned odor. Finally, there were no respiratory differences in response to the learned odor during the neurophysiological recordings (Wilson et al., 1987). These data suggest that there may be changes intrinsic to the bulb that produce the specific, altered output signal by the bulb in response to conditioned odors.

The signal from the bulb in response to learned odors is therefore reflected in a decrease, rather than an increase, in neural activity. Olfactory projection areas must therefore be able to recognize a decrease in ongoing activity from the bulb to identify the learned odor.

In addition to the periglomerular suppression of mitral responses, there is also the possibility that a subset of tufted cells acts to suppress neighboring mitral cell activity in response to a conditioned odor. If glomerular-layer tufted cells were increasing their activation of granule cells via their axon collaterals, then one might observe increased activity in that region in response to the learned odor. To obtain the resolution required to study this possibility, we developed a stain for glycogen phosphorylase in the bulb, a stain previously shown to be activity dependent ( Woolf, Chong, and Rashdi, 1985). We found a restricted patch of activity in the granule cell layer/internal plexiform layer of trained pups when they were exposed to the trained odor (Coopersmith and Leon, 1987). Both periglomerular and tufted cell modifications may therefore play a role in altering the output signal from the bulb in response to conditioned odors.

Neuroactive Agents and Early Olfactory Learning

Exitatory Amino Acids. The olfactory system has a large number of neuroactive agents and neuro- modulators that are either intrinsic to it or project to it. We have studied the role of one neuroactive agent that appears to be involved in neurotransmis- sion from the bulb and one involved in centrifugal transmission to the bulb in the development of the neurobehavioral response to conditioned odors.

The putative neurotransmitters glutamate and N-aspartylglutamate appear to be present in the mitral and tufted cells of the olfactory bulb (Saito, Kumoi, and Tanaka, 1986; Anderson, Henderson, Cangro, Namboodiri, Neale, and Cotman, 1987 ), possibly activating the dendrites of granule cells

within the bulb (Shepherd, 1972). Glutamate receptors of the N-methyl-D-aspartate (NMDA) type are present in the external plexiform layer of neonatal rat olfactory bulbs (Lincoln, Coopersmith, Harris, Cotman, and Leon, 1986) as well as the main projection sites of the bulb, including the pir- iform cortex (Monaghan and Cotman, 1985). To determine whether activation of these receptors plays a role in the development of the neurobehavioral response to early olfactory conditioning, we preceded each odor/stroking pairing on PND 1 - 18 either with or without an injection of 2-APV to block the NMDA receptors.

Those rats receiving the drug developed neither the behavioral preference nor the enhanced glomerular-layer response to the trained odor that was characteristic of saline control pups (Lincoln, Coo- persmith, Harris, Cotman, and Leon, 1988). Simi- lar data has recently been presented showing the dependence upon these receptors for early aversive conditioning (Stanton and Jensen, 1988). They went on to report no primary deficit in either anos- mia or motor behavior that was underlying this deficit after drug injection. While further work needs to determine the specificity of this phenomenon, the data are consistent with the notion that early olfactory learning requires activation of these receptors.

Activation of NMDA receptors has recently been shown to affect neuronal process outgrowth and branching ( Pearce, Cambray-Deakin, and Burgoyne, 1987; Brewer and Cotman, 1989). Be- cause dendritic arborization could participate in an increase in glomerular size in trained pups, NMDA activation might be involved in producing the structural changes observed in the bulb as a consequence of early olfactory learning.

Norepinephrine. The locus coeruleus sends a major centrifugal input to the olfactory bulb, attract- ing about 40% of the noradrenergic fibers (Shipley, Halloran, and De la Torre, 1985). These projections are present (McLxan and Shipley, 199 1 ) and functional (Wilson and Leon, 1988b) within the first week of life. Importantly, the tactile stimulation used during early olfactory conditioning evokes a clear response by locus coeruleus neurons as early as PND I (Nakamura, Kimura, and Saka- guchi, 1987).

When the P-noradrenergic blocker propranolol was injected before the daily odor/ tactile stimulation pairings on PND 1-18, it blocked the behavioral preference, the enhanced 2-DG uptake in the glomerular foci, and the suppressed neurophysio-

1566 Leon

logical response by the output neurons (Sullivan et al., 1989a). In addition, when the tactile stimulation was replaced by the /3-noradrenergic agonist isoproterenol, pups developed the behavioral, 2- DG, and neurophysiological responses characteristic of odor/tactile stimulated controls. Moreover, the dose-response curve for isoproterenol described the inverted U-shaped function typical of noradrenergic facilitation of learning in adults ( McGaugh, 1983 ).

The locus coeruleus may be specially adapted to mediate the stimulation experienced by pups in the nest. In early life, the locus coeruleus responds vigorously to either noxious stimulation or tactile stimulation of the sort we have been discussing (Nakamura et al., 1987). Nonnoxious tactile stimuli become much less able to evoke a locus coeruleus response in adulthood. In addition, the locus coeruleus neurons appear to be electronically coupled in neonates, a phenomenon that decreases after PND 10 (Christie, Williams, and North, 1989). Such coupling could increase the sensitivity of the locus coeruleus to tactile stimulation by increasing the number of neurons activated by a particular stimulus.

These data raise the interesting possibility that noradrenaline could be the final common pathway for the variety of stimuli that can reinforce early olfactory learning. These stimuli include: stroking (Pedersen, Williams, and Blass, 1982; Sullivan, Brake, Hofer, and Williams, 1986a; Sullivan, Hofer, and Brake, 1986b); milk (Johanson and Teicher, 1980; Brake, 1981; Johanson and Hall, 1982; Johanson, Poleferone, and Hall, 1984; Sulli- van and Hall, 1988); warmth (Alberts and May, 1984), suckling ( Amsel, Burdette, and Letz, 1976; Kenny, Stoloff, Bruno, and Blass, 1979); and tail- pinch (Sullivan et al., 1986a). All these stimuli would be expected to be experienced within the nest and may also all stimulate the locus coeruleus early in life. It also seems possible that long-term isolation paired with odor exposure (Leon et al., 1977 ) also was accompanied by an increase in noradrenergic activity, thereby allowing the observed olfactory preference to be formed.

While there is almost certainly a direct action of noradrenaline on neural activity in the bulb, there may also be another route by which it may modu- late neural activity in that structure. We recently found that glycogen levels are extremely high in the olfactory bulb (Coopersmith and Leon, unpublished observations) and that glycogen phosphorylase, the rate-limiting enzyme mobilization of glycogen to glucose, is similarly high in that structure

(Coopersmith and Leon, 1987). Noradrenaline mobilizes glucose in the brain via its action on glycogen phosphorylase ( Edwards, Nahorski, and Rogers, 1973). IJsing an in vitro olfactory slice preparation, we found that noradrenaline, even at low levels, rapidly induces the mobilization of glucose from glycogen in the bulb ( Coopersmith and Leon, 1988b). Glucose has been shown to mimic the facilitatory erects of norepinephrine on memory (Gold, Vogt, and Hall, 1986), and it is possible that during early olfactory learning the critical role of noradrenaline involves the mobilization of glucose.

COMPARATIVE ISSUES

Comparative Analysis of Rodent Neurobehavioral Development

Mammals are born on a precocial-altricial continuum, their independence from maternal care vary- ing greatly even among rodents. One extreme on this continuum is the spiny mouse (Acomys cahirinus), unique among muridae in that it is born sighted, furred, and mobile (von Dieterlen, 1962). Much less mature at birth are the Norway rats (Rattus norvegicus) we have been discussing. These rats are born sightless, nude, thermally unsta- ble and have poor mobility (Adolph, 1957; Altman and Sudarshan, 1975; Taylor, 1960). Even less mature than Norway rats at birth are Mongolian gerbils (Mer ion t~ unguiculatus), whose capacities develop slowly ( McManus, 197 1 ; Kaplan and Hy- land, 1972).

All three species are attracted to the parental odors, (Leon and Moltz, 197 1 ; Porter and Ruttle, 1975; Gerling and Yahr, 1982) although the temporal pattern of the onset and termination of attraction to such odors differs among them. Specifi- cally, spiny mice start to approach their mother’s odor within the first 26-36 h after they are born and cease approaching the odor by PND 25 (Porter and Ruttle, 1975; Porter and Doane, 1976; Porter, Doane, and Cavallero, 1978). Recall that Norway rats begin to approach their mother’s odor at PND 12-14 and cease their attraction by PND 27 (Leon and Moltz, 1972). Gerbils begin to be attracted to their mother’s odor at 3 weeks postpartum, continue to be attracted to the odor at 6 weeks, but fail to approach it at 9 weeks (Yahr and Anderson- Mitchell, 1983).

We found a striking correlation between the time at which the young of all three species start to

Olfclctory Prejkrence Development 1567

approach their mother’s odor and the time when their olfactory bulbs reach an adult level of structural organization (Leon et al., 1984). What this correlation means is unclear; clearly, these animals are able to perceive odors before they approach the dam. Moreover, their ability to approach the odors is independent of the onset of maternal odor emission (Altman and Sudarshan, 1975; Leon and Moltz, 1972; Porter et al., 1978). The maturation of the olfactory system therefore may signal the end of early plasticity and/or be correlated with maturational changes elsewhere in the brain that stimulate approach to the odor from a distance.

The time of termination of the approach response correlates with the time of weaning in each species. While spiny mice are capable of being weaned at PND 6, they are normally nurtured by their mother until about 4 weeks postpartum, the time at which normally cease their responsiveness to the odor (Porter, 1983). Norway rat pups cease their approach to the odor at about the time of weaning, at the end of the fourth postnatal week ( Leon and Moltz, 1972). While gerbil pups can be weaned after about 4 weeks, they normally over- winter with their parents (Bannikov, 1954; Kaplan and Hyland, 1972; Thiessen and Yahr, 1977). It seems likely that they use their olfactory attraction to maintain proximity with their parents through that prolonged period.

Role of Olfaction in Mother-Young Relationships in Pallid Bats

When mother rodents return to the litter within the maternal nest, they are reasonably well assured that they will be caring for their own offspring. Other species return to nest sites in which the young of the offspring of other mothers are present. Here, a central problem for mothers is to identify their own offspring before investing their lactational resources in the growth of the young of other mothers. We therefore investigated the role of olfaction in facilitating the identification of the young by the mother. We then made the first steps in identifying a neural response to pup odors.

Juvenile bats living in temperate climates must huddle if they are to survive in the absence of their mothers (Beasley and Leon, 1986). Therefore, when their mothers return from their extensive for- aging they must discriminate and approach only their own young if they are to maximize their re- productive investment. This problem is in particular dramatic in a species such as the Mexican free- tailed bat ( Tadarida braziliensis), which nests in

colonies numbering in the millions. Even under such conditions, individual mothers seek out and nurse their own young with surprising regularity, as indicated by allozyme analysis ( McCracken, 1984 ) . Indeed, mother bats nurse only their own young in almost all species of bats that have been studied (Bradbury, 1977).

Pallid bats (Antrozous pallidus) are insectivor- ous vespertilionid bats found throughout western North America. Colony females synchronously give birth to two pups each and the mothers cluster in groups of 20-200 during lactation (Brown, 1976). Pallid bat pups start to fly at 4-5 weeks and are weaned at 7-10 weeks (Brown, 1976). While the mother forages for insects, the young are left in a creche that allows them to retain heat sufficiently to survive their mother’s absence (Beasley and Leon, 1986). Returning mothers, however, are able to identify and nurse their own young (Brown, 1976). One mechanism used for mother-young identification is a signature call that each pup can emit that elicits a response by their mother (Brown, 1976 ).

Olfactory cues may also play a role in the final identification of the young, as pallid bat mothers sniff their young carefully before nursing is allowed (Brown, 1976). In fact, Mexican free-tailed bat mothers will preferentially approach the odor of their own pup (Gustin and McCracken, 1987), and recognition of pups is due to both maternal scent marking of the young and odor production by the pups themselves (Loughry and McCracken, personal communication).

We first tested differential behavioral responses to maternal odors by pups by monitoring their respiration in an airtight compartment. We found no differences between their respiration to maternal odors and fresh air. On the other hand, mother bats had a differential respiratory response to the odor of their own young when compared to that of a strange pup (Fig. 5 ) .

We then looked to see whether mothers had a differential olfactory bulb response to the odor of their pups. Mothers exposed to the strange pup odor had increased 2-DG uptake within a unique macroglomerular structure on the dorsomedial- ventromedial aspect of the bulb relative to that shown for their own pups (Fig. 6; Beasley, Coo- persmith, and Leon, 1986). This macroglomerular structure has one to three layers of glomeruli clus- tered together and may be a specialized structure to process species-typic chemical cues in pallid bats. The increased respiration by mothers to the odor of a strange pup may be the cause of the increased

1568 Leon

8000 I 1 I - I

I

Mother Bat to Strange Pup Odor

Figure 5 Respiratory behavior of mother pallid bats to the odor of either their own or a strange pup.

2-DG uptake within the macroglomeruli. Because all the pups appear to attempt to attach to the nip- ples of any passing mother, the critical response of mothers may be to dissuade strangers from attach- ing rather than soliciting attachment by their own. In that case, the increased behavioral and neural responsiveness to strange pup odor may thereby be explained.

These data also raise the possibility that mother bats may have a plasticity similar to neonatal rat brains in response to learned odors. Indeed, we have some evidence suggesting that adult bats may have neotenized brains. Specifically, the cortical or-

ganization of commissural connections in adult bats closely resembles that of neonatal rodents (Ivy, Beasley, Leon, and Lynch, 1985). Moreover, bats are both extremely long lived (Tuttle and Ste- venson, 1983) arid have low brain levels of calpain, a calcium-activated protease that may be involved in cellular aging (Lynch and Baudry, 1984; Baudry, DuBrin, Beasley, Leon, and Lynch, 1986). The lack of calpain in bat brains may slow their aging and thereby allow a neural plasticity in adults that is not possible in the brains of other mammals. The ability of mother bats to develop a neurobehavioral response to their young each year may reflect such a plasticity.

Development of Learned Olfactory Preferences in Human Infants

Human infants come to prefer the odor of their mother over the course of the first few days of life (Macfarlane, 1975; Russell, 1976; Schaal, 1988). Infants have also been shown to develop a preference for a nonmaternal odor (Balogh and Porter, 1986) but it was unclear whether such learning was due to an associative process or simply a matter of odor exposure. We therefore determined whether odor exposure concurrent with tactile stimulation could facilitate olfactory preference formation in PND 1 human infants (Sullivan, Taborsky-Bar-

. 0 own pup odor to T

T

T

Dorsomedial Ventromedial Ventrolateral Dorsolateral Area of Ol factory Bulb ( 7 0 0 - 1 0 0 0 p m fr'om rostrol pole)

Figure 6 2-DG uptake in a macroglomerular complex found in a region 700-1000 pm from the rostra1 pole of the bulb of mother pallid bats in response to either the odor ofeither own or a strange pup.


bar, Mendoza, Itino, Leon, Cotman, Payne, and Lott, 1991). On the first day of life, babies were presented with an odor paired with a firm but gen- tle tactile stimulation.

On the second day of life, infants were tested for an olfactory preference that could be evidenced by a head turn toward the odor or a conditioned increase in body movement. Infants receiving concurrent odor and tactile stimulation demonstrated both a conditioned head turn and a conditioned increase in body movement to the trained odor. Those babies that received sequential exposure to the tactile stimulation and the odor developed neither conditioned response. Neither did babies exposed to odor alone alter their response to the trained odor. The conditioned responses were also shown to be odor specific, indicating that the heightened response to the learned odor did not reflect a generalized increase in odor responsiveness. These data bear a remarkable similarity to those seen for rats and suggest that both species can acquire olfactory preferences early in life via classic conditioning.

CONCLUSION

Similarities between this work and other research on developing mammalian brains raise the fascinat- ing possibility that there may be mechanisms of neural organization that are shared throughout the brain. Specifically, there are parallels emerging between this research and that dealing with the estab- lishment of ocular dominance within the visual system of developing cats. Visual stimulation, nec- essary to maintain binocularity of striate cortical neurons, is only effective in influencing cortical organization if it is accompanied by nonspecific stimulation from the thalamus or reticular formation (Singer and Rauschecker, 1982). These data are reminiscent of the finding that nonspecific tactile stimulation (possibly activating the same brain areas) is needed for early olfactory learning (Sulli- van and Leon, 1986). Another similarity has been observed in visual, olfactory, and somatosensory developmental plasticity: All seem to require noradrenergic stimulation (Bear and Singer, 1986; Sullivan and Leon, 1986; Kasamatsu and Petti- grew, 1979; Loeb, Chang, and Greenough, 1986). Finally, blocking NMDA-type glutamate receptors interferes with both ocular dominance plasticity (Kleinschmidt, Bear, and Singer, 1987) and the neurobehavioral consequences of early olfactory preference training (Lincoln et al., 1988).

We have shown that mother rats produce odors that attract their young and that this type of experience produces changes in the anatomy and function of the bulb to such odors. This critical type of learning appears to evoke large and permanent neural changes that make the system amenable to experimental analysis. The developing mammal learns about a few important aspects of its environment critical for survival and the study of the neural basis for these simple, but important, neurobehavioral changes can reveal a great deal about the mechanisms that govern the early lives of indi- viduals.

The research described above was supported in part by Grant HD42236 from NICHD, Grant NO0014-89-J- 1960 from ONR, and Research Scientist Development Award 0037 1 from NIMH.

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Neuroethology of olfactory preference development

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