Developmental Brain Research, 12 (1984) 45-53 45 Elsevier

Development of Olfactory Bulb Organization in Precocial and Altricial Rodents

MICHAEL LEON l, ROBERT COOPERSMITH 1, CATHERINE ULIBARRI 1, RICHARD H. PORTER 2 and J. BRADLEY POWERS 3

t Department of Psychobiology, University of California, Irvine, CA 92717, 2John F. Kennedy Center for Research on Education and Human Development, George Peabody College, Nashville, TN37203, and 3Department of Psychology,

Vanderbilt University, Nashville, TN, 37240 (U.S.A.)

(Accepted October 4th, 1983)

Key words: olfactory bulb development - - precocial-altricial development - - maternal chemoattractants - - Norway rats --Mongolian gerbils - - spiny mice

The structural organization of the olfactory bulbs of spiny mice, Norway rats and Mongolian gerbils was followed over the course of their development. The pups of all 3 species normally begin to approach the odor of their dams at a time when their olfactory bulbs are at a similar stage of development. The data suggest that there may be a common aspect of olfactory bulb development that underlies the onset of olfactory guided approach behavior in rodents.

INTRODUCTION

Rodents are born on a precocial-altricial contin-

uum, with their relative independence from their

dams varying greatly across species. At one extreme is the spiny mouse (Acomys cahirinus), unique

among muridae in that it is born furred, sighted and mobile 13. Somewhat more retarded in their devel-

opment are Norway rat young (Rattus norvegicus), who are born nude, sightless and unable either to

thermoregulate effectively, or to move away from

the confines of the maternal nest. Fur, thermoregula-

tory capacity, sight and mobility develop during the third week postpartum1,3.9,53. Even more retarded in

their development are Mongolian gerbils (Meriones unguiculatus), who also are born nude and blind, who thermoregulate poorly, and who are unable to move away from the nest 29,3t. The auditory, visual and mo-

tor systems develop even more slowly in this species than in Norway rats15,22, 29.

In all 3 species, the mother and young must be re- united periodically for nursing episodes, and such in- teractions may well be facilitated by an attraction to maternal odors that has been found for the young of

all 3 species16,25,27, 45. However, the onset and termi-

nation of the attraction to the maternal odor differ

among the species. Spiny mice begin to approach the

odor of their dam within the first 26-36 h after their birth 40,45 ceasing to be attracted to the odor by day 25

postpartum 42. Norway rat young begin to approach to odor of their dam beginning at days 12-14 postpar-

tum and continue to be attracted to that odor until the

end of the fourth postpartum week 28. Gerbils begin

to be attracted to the odor of their dam at 3 weeks

postpartum, continue to be attracted at 6 weeks, but cease to be attracted to the odor at 9 weeks 58 (but see ref. 12).

One explanation for the differences in the timing

of the olfactory-based approach responses of the

young is that the mothers differ in the onset and ter- mination of their odor emission. However, the peri- od during which the pups approach the dam is corre-

lated with, but independent of, the period during which the dams emit the attractant32,42.

The second possibility is that differences in motor development underlie differences in the onset of ap- proach to the odor. The young may be attracted to the odor, but may not be able to express that attrac-

"Correspondence: M. Leon, Department of Psychobiology, University of California, Irvine, CA 92717, U.S.A.

46

tion until their motor systems are sufficiently devel- oped to allow them to approach the odor source.

Spiny mice, for example, are mobile at birth and ap-

proach the odor of their dam on day 1 postpartum, while Norway rat pups are not particularly mobile

until the beginning of the third postpartum week, when they begin to approach the maternal odor 4,9,28.

On the other hand, Norway rats and gerbils are capa- ble of directed movement early in life ~2.23, suggesting

that there may be other factors involved in eliciting

the onset of olfactory guided approach behavior.

A third possibility is that there are neural changes in the olfactory systems of these 3 species that under- lie the behavioral responsiveness of the young to the

odor of the dam. If that is the case, one might expect there to be a comparable stage of olfactory system development across the 3 species at the time of the onset of attraction to maternal odors. We therefore examined the structural changes in the olfactory bulbs of the young in the 3 species and attempted to correlate their neural development with their devel- opment of approach behavior to maternal odors.

MATERIALS AND METHODS

Spiny mouse dams and their litters from a colony at the John F. Kennedy Center in Nashville were kept at 23 + 2 °C and lights remained on between 06.00 and 19.00 h. The mothers had constant access to Pu- rina Mouse Chow and tap water. On both day 1 and on day 14 postpartum, 5 pups were sacrificed, per-

fused with saline and formalin, and the olfactory bulbs were removed. To facilitate the sectioning of the small bulbs, the tissue was then embedded in par- affin. Coronal sections of the tissue were cut at 30/~m, and every 10th section was stained with

Azure A. Norway rat dams were obtained while pregnant

from Simonsen Breeders (Gilroy, CA). These dams and their young were kept at 21 + 1 °C and lights re- mained on from 08.00 to 18.00 h. There was constant access for the mothers to Wayne Lab Blocks and tap water. On days 1,7, 14, 21 and 28 postpartum, 2 pups were decapitated, and their olfactory bulbs were quickly removed. The tissue then was frozen at -15 °C and 30/~m coronal sections were then cut on a cryostat. The tissue was then mounted and stained

with Azure A.

Mongolian gerbil dams, originally obtained as vir- gins from Tumblebrook Farms (West Brookfield, MA), were kept with their litters at 17 + 1 °C, with

the lights on between 06.00 and 20.00 h. The dams had constant access to Wayne Lab Blocks and tap

water. On days 1, 7, 14, 21, 28, 35, 42, 49 and 56, two pups had their olfactory bulbs removed, frozen, cut

and stained as described for the Norway rats.

RESULTS

Figs. 1-4 show clear differences in the devel-

opment of olfactory bulb organization among the 3 species. On day 1, spiny mice have what appears to be a mature olfactory bulb. The glomeruli are dis- tinct, the mitral cells are arranged in a single row, and the granule cell layer is clearly evident. There is no obvious further differentiation occurring over the next 2 weeks postpartum (see Figs. 1 and 4).

The development of Norway rat olfactory bulb or- ganization has been described in substantial de- tail2, 47,51. On day 1, the glomeruli and mitral laminae

are evident, though the mitral cells are arranged 2-3 deep (see Figs. 2 and 4). There is also a relatively

wide internal plexiform layer and a limited granule cell population which is still concentrated in the sube- pendymal surround. By day 7, the granule cells have begun to migrate to the granule cell layer in what ap- pear to be clusters of cells. This pattern of migration may reflect families of dividing granule cells. On day 14, the mitral cells are arranged in a single row, most of the granule cells are present, maintaining their clustered organization, and there is a narrowing of the internal plexiform layer. There is relatively little further change in olfactory bulb organization through day 28. It is at day 14 that the rat olfactory bulb matches the maturity of the day 1 spiny mouse.

The gerbil bulb is a largely undifferentiated struc- ture on day 1. The laminae can barely be discerned, the mitral cells are arranged in a row 2-3 deep, and the granule cell layer has not been differentiated (see Figs. 3 and 4). It is not until day 7 that the organiza- tion resembles that of the day 1 rat. The bulb appears to mature by day 21, at which time it is comparable to the development of the day 1 spiny mouse and the day 14 rat. No further change in gerbil olfactory bulb organization is observed through day 56.

The developmental sequence of the gerbils in- volves the thinning of the mitral cell layer and the narrowing of the internal plexiform layer relative to the external plexiform layer. This sequence is similar to that seen in the rats, but appears to lag 1 week be- hind the rats. Thus, in day 1 rats and day 7 gerbils, the width of the internal plexiform layer is comparable to that of the external plexiform layer. It is not until day 14 in rats and day 21 in gerbils that the relative sizes of these layers are seen as they appear in the adult. A similar pattern is seen for the thinning of the mitral cell layer; the single cell layer characteristic of the adult is seen by day 14 in rats and not until day 21 in gerbils. It should be noted that both of these features are mature in the day 1 spiny mouse.

DISCUSSION

There is a striking correlation between the time at which the young rodents start to approach the odor of their mother and the time at which the bulb reaches an adult level of structural organization. Reliable ap- proach to maternal odors is observed in day 1 spiny

47

mice, day 14 Norway rats and day 21 gerbils28,45, 58,

just at the times that their olfactory bulbs reach a ma- ture structural organization.

One explanation for this relationship may be that the olfactory system must be completely mature be- fore it can sense odors. The available evidence, how- ever, suggests that that is not the case. For example, each type of olfactory bulb connection has formed by the time of birth in laboratory mice, although these connections increase in number during devel- opment tT. In Norway rats, the afferents from the bulb also are present at birth, also increasing postna- tally49, 57. Neural activity patterns in the bulb of neo-

natal rats, measured by uptake of radiolabeled 2-de- oxyglucose uptake, are qualitatively, though not quantitatively, similar to adult uptake patternsS,6,55. Moreover, the first suckling response of Norway rats is mediated by olfactory cues 54, the stimulus rele- vance of which appears to be acquired perina- tally 36,37. Young rats also orient to the maternal odor before they begin to approach it 4. It is therefore un- likely that the relative immaturity of the bulb pre- vents approach behavior by preventing processing of

SPINY MOUSE

Fig. 1. Coronal sections of spiny mouse olfactory bulbs on days 1 and 14 postpartum.

48

RAT

Fig. 2. Coronal sections of rat olfactory bulbs on days 1, 14, 21 and 28.

GERBIL

49

Fig. 3. Coronal sections of gerbil olfactory bulbs on days 1,21,28 and 56.

5(]

GERBIL RAT

< Q

!

Q

N ¸

<

Fig. 4. Coronal sections of the olfactory bulbs of gerbils, rats, and spiny mice at a high magnification. Black bordered sections indicate the age at which olfactory guided approach behavior appears in each species. GL, glomerular layer; M, mitral cell layer: Gr, granule cell layer; EPL, external plexiform layer; IPL, internal plexiform layer.

51

maternal odors. A second possibility is that the olfactory system is

mature enough to allow approach, but that the moti-

vational systems underlying approach are not. In- deed, motivational status influences mitral cell firing rates via centrifugal connections in rats 33-35. The mo-

tivation in the case of rat pup approach behavior ap- pears to involve avoidance of unfamiliar stimuli, a re- sponse pattern that begins at about the end of the sec-

ond week. This phenomenon is evidenced by the fact that pups deprived of familiar maternal odors have a striking increase in locomotion that is suppressed if the odor is present 10,11.18-21,28,46. Their behavior in

the absence of the maternal odor may be seen as es- cape from an unfamiliar surround, for if the surround is made familiar through daily experience, the pups are less likely to approach the familiar maternal odors 28. The brain areas mediating the responsive-

ness to unfamiliar surroundings may mature at the same time that the olfactory bulbs mature. The matu- ration of these areas may thereby allow the young to begin to approach the maternal odor.

A third possibility involves the fact that all 3 spe- cies seem to acquire their responsiveness to maternal odors principally through postnatal experience. The dominant maternal odors in spiny mice, rats and probably gerbils can differ among mothers, as they are dependent on maternal diet; the pups approach those maternal odors with which they are famil- iar 14,16,24,26,39,41,52. In fact, rats and spiny mice will

even approach an arbitrarily selected odor if exposed to it early in life 26,43,44.

This plasticity may lie in the olfactory system, and may be possible only during early life, perhaps be- cause the inhibitory interneurons - - the granule cells

- - are not yet present and cannot modulate mitral cell firing as they do in adulthoodSO. The unique neo- natal firing patterns of the mitral cells 30 may allow ol- factory cues to acquire a special status for the young. The changes underlying this plasticity may be com- pleted only at the time of olfactory bulb maturation.

In Norway rats, the electrical activity of the olfac- tory bulb reaches adult patterns at 12 days postpar- tum 48, the time when the granule cell population pla- teaus 2, when the laminae are largely mature and about the time when the rats begin to approach their damS. It is possible that the maturation of the granule cell population reduces the plasticity of the olfactory

system, and thereby focuses the young on the domi-

nant odors with which they have had significant expe-

rience. The young would then begin to focus their ap- proach on the maternal odor. Young rats with a ma-

jor portion of their granule cell population destroyed by X-irradiation still approach maternal odors 59, but it remains possible that relatively few granule cells

are required to mediate this process. Termination of approach behavior to maternal ol-

factory cues seems to occur when the young are being

weaned and when the olfactory system has attained an adult level of organization. While spiny mouse pups occasionally nurse for 4 weeks postpartum, they can be weaned as early as 6 days of age 38. By 25 days postpartum, they no longer approach the maternal odor even though the dams continue to emit it 4°,42.

Young spiny mice apparently can be independent of the mother shortly after birth, but maintain nurtural interactions with her. Olfactory attractants may be

particularly important in a species in which both mother and young are mobile, and the duration of their interactions is variable.

Norway rat pups stop approaching the maternal odor at the end of the fourth week, when their dams stop emitting the odor 28, the pups are being weaned 7

and olfactory bulb electrical activity resembles that of the adult 48. These olfactory cues may tether the pups to the nest site while they are mobile but still de- pendent on periodic contact with their dam.

While gerbil pups can be weaned in the fourth postpartum week 22,56, they normally overwinter with

their parents s. It seems likely that they use olfactory cues to maintain proximity with their parents over that extended period.

We have demonstrated a clear relationship be- tween the development of the structural organization of the olfactory bulb and the development of ap- proach behavior to maternal odors in 3 species of ro- dents. The mechanisms underlying the development of olfactory-guided behavior in young rodents will be addressed in future research.

ACKNOWLEDGEMENTS

This research was supported by NSF Grant BNS 80-23107 to M.L., who holds Research Scientist De- velopment Award MH00371 from A D A M H A , NIMH, NIMH Grant MH-26481 to Pauline Yahr,

52

N I C H D g ran t 00973 to R . H . P . , a n d N I H G r a n t

H D 1 4 5 3 5 to J . B . P . W e t h a n k H e r b Ki l l ackey for his

adv ice and Pau l i ne Y a h r for he r c o m m e n t s on the

m a n u s c r i p t .

REFERENCES

1 Adolph, E, F., Ontogeny of physiological regulations in the rat, Quart. Rev. Biol., 32 (1957) 89-135.

2 Altman, J. and Das, G. D., Autoradiographic and histolog- ical studies of postnatal neurogenesis. I. A longitudinal in- vestigation of cells incorporating tritiated thymidine in neo- nate rats, with special reference to postnatal neurogenesis in some brain regions, J. comp. Neurol., 126 (1966) 337-39O.

3 Altman, J. and Sudarshan, K., Postnatal development of locomotion in the laboratory rat, Anita. Behav., 23 (1975) 896-920.

4 Airman, J., Sudarshan, K., Das, G. D., McCormick, N. and Barnes, D., The influence of nutrition on neural and behavioral development: III. Development of some motor, particularly locomotor patterns during infancy, Develop. P~ychobiol., 4 (1971) 97-114.

5 Astic, L. and Saucier, D., Ontogenesis of the functional ac- tivity of rat olfactory bulb: autoradiographic study with the 2-deoxyglucose method, Develop. Brain Res., 2 (t982) 243-256.

6 Astic, L. and Saucier, D., Metabolic mapping of functional activity in the olfactory projections of the rat: ontogenetic study, Develop. Brain Res., 2 (1982) 141-156.

7 Babicky, A., Ostadalova, I., Parizek, J., Kolar, J. and Bibr, B., Use of radioisotope techniques for determining the weaning period in experimental animals, Physiol. Bo- hemoslov., 19 (1970) 457-467.

8 Bannikov, A. G., The places inhabited and natural history of Meriones unguiculatus. In Mammals of the Mongolian Peoples Republic, USSR Acad. Sci., 1954, pp. 410-415 (translation by D. Lay, provided by Tumblebrook Farms).

9 Bolles, R. C. and Woods, J., The ontogeny of behavior in the albino rat, Anim. Behav., 12 (1964) 427--441.

10 Campbell, B. A., Lytle, L. D. and Fibiger, H. C., Ontoge- ny of adrenergic arousal and cholinergic inhibitory mech- anisms in the rat, Science, 166 (1969) 637--638.

11 Campbell, B. A. and Raskin, L. A., Ontogeny of behavior arousal: the role of environmental stimuli, J. comp. physi- ol. PsychoL, 92 (1978) 176-184.

12 Cornwell-Jones, C. A. and Azar, L. M., Olfactory devel- opment in gerbil pups, Develop. Psychobiol., 15 (1982) 131-137.

13 von Dieterlen, F., Geburt and Geburtshilfe bei der Stachel- maus Acomys cahirinus, Z. Tierpsychol., 19 (1962) 191-222.

14 Doane, H. M. and Porter, R. H., The role of diet in mother- infant reciprocity in the spiny mouse, Develop. Psycho- biol.. 11 (1978) 271-277.

15 Finck, A., Schneck, C. D. and Hartman, A. F., Devel- opment of cochlear function in the neonate Mongolian ger- bil (Meriones unguiculatus), J. comp. Physiol. Psychol., 78 (1972) 375-380.

16 Gerling, S. and Yahr, P., Maternal and paternal phero- mones in gerbils, Physiol. Behav., 28 (1982) 667-673.

17 Hinds, J. W. and Hinds, P. L., Synapse formation in the mouse olfactory bulb, J. comp. Neurol.. 169 (1976) 15-40.

18 Hofer, M. A., Maternal separation affects infant rat's be- havior, Behav. Biol., 9 (1973) 629-633.

19 Hofer, M. A., The role of nutrition in the physiological and behavioral effects of early maternal separation on infant rats, Psychosom. Med., 35 (1973) 350-359.

20 Hofer, M. A., Studies on how early maternal separation produces behavioral change in young rats, Psychosom. Med., 37 (1975) 245-264.

21 Hofer, M. A., Olfactory denervation: its biological and be- havioral effects in infant rats, J. comp. Physiol. Psychol., 90 (1976) 829-838.

22 Kaplan, H. and Hyland, S. O., Behavioral development in the Mongolian gerbil (Meriones unguiculatus), Anim. Be- hay., 20 (1972) 14%154.

23 Kleitman, N. and Satinoff, E., Thermoregulatory behavior in rat pups from birth to weaning, Physiol. Behav., 29 (1982) 537-541.

24 Leon, M., Dietary control of maternal pheromone in the lactating rat, Physiol. Behav., 14 (1975) 311-319.

25 Leon, M., Filial responsiveness to olfactory cues in rats. In J. S. Rosenblatt, R. A. Hinde and M. C. Busnell, (Eds.), Advances in the Study of Behavior, Vol. VIII, Academic Press, New York, 1978, pp. 11%153.

26 Leon, M., Galef, B. G. and Behse, J., Establishment of pheromonal bonds and diet choice in young rats by odor pre-exposure, Physiol. Behav., 18 (1977) 387-391.

27 Leon, M. and Moltz, H., Maternal pheromone: Discrimi- nation by preweanling albino rats. Physiol. Behav., 7 (1971) 265-267.

28 Leon, M. and Moltz, H., The development of the pheromo- nal bond in the albino rat, Physiol. Behav., 8 (1972) 68,3-686.

29 Marston, J. H. and Chang, M. C., Breeding management and reproductive physiology of the Mongolian gerbil (Mer- iones unguiculatus), Lab. Anita. Care, 15 (1965) 34-48.

30 Math, F. and Davrainville, J. L., Electrophysiological study on the postnatal development of mitral cell activity in the rat olfactory bulb, Brain Res., 190 (1980) 243-247.

3l McManus, J. J., Early postnatal growth and the devel- opment of temperature regulation in the Mongolian gerbil ( Meriones unguiculatus), J. Mammal., 52 (1971) 782-792.

32 Moltz, H. and Leon, M., Stimulus control of the maternal pheromone in the lactating rat, Physiol. Behav., 10 (1973) 69-71.

33 Pager, J., A selective modulation of the olfactory bulb elec- trical activity in relation to the learning of palatibility in hungry and satiated rats, Physiol. Behav.. 12 (1974) 189-195.

34 Pager, J., A selective modulation of olfactory input sup- pressed by lesions of the anterior limb of the anterior com- missure, Physiol. Behav.. 13 (1974) 523-526.

35 Pager, J., Giacheni, I., Holley, A. and LeMagnen, J., A selective control of olfactory bulb electrical activity in rela- tion to food deprivation and satiety in rats, Physiol. Behav., 9 (1972) 573-579.

36 Pederson, P. E. and Blass, E. M., Prenatal and postnatal determinants of the first suckling episode in albino rats, De- velop. Psychobiol.. 15 (1982) 34%356.

37 Pederson, P. E., Williams, C. L. and Blass, E. M., Classical conditioning of suckling behavior in three day old albino rats, J. exp. Psychol., 8 (1982) 329-341.

38 Porter, R. H., Communication in rodents: adults to infants. In R. W. Elwood (Ed.), Parental Behavior of Rodents. John Wiley, New York, 1983, pp. 95-125.

39 Porter, R. H., Deni, R. and Doane, H. M., Responses ot Acornys cahirinus pups to chemical cues produced by a fos- ter species, Behav. Biol., 20 (1977) 244-251.

40 Porter, R. H. and Doane, H. M., Maternal pheromone in the spiny mouse (Acomys cahirinus), Physiol. Behav., 16 (1976) 75-78.

41 Porter, R. H. and Doane, H. M., Dietary-dependent cross- species similarities in maternal chemical cues, Physiol. Be- hay., 19 (1977) 129-131.

42 Porter, R. H., Doane, H. M. and Cavallaro, S. A., Tempo- ral parameters of responsiveness to maternal pheromone in Acomys cahirinus, Physiol. Behav., 21 (1978) 563-566.

43 Porter, R. H. and Etscorn, F., Olfactory imprinting result- ing from brief exposure in Acomys cahirinus, Nature (Lond.), 250 (1974) 732-733.

44 Porter, R. H. and Etscorn, F., Primary effect for olfactory imprinting in the spiny mouse (Acornys cahirinus), Behav. Biol., 15 (1975) 515-517.

45 Porter, R. H. and Ruttle, K., The responses of one-day old Acomys cahirinus pups to naturally occurring chemical stimuli, Z. Tierpsychol., 38 (1975) 154-162.

46 Randall, P. K. and Campbell, B. A., Ontogeny of behavior- al arousal in rats: effect of maternal and sibling presence, J. comp. physiol. Psychol., 90 (1976) 453-459.

47 Rosselli-Austin, L. and Altman, J., The postnatal devel- opment of the main olfactory bulb of the rat, J. develop. Physiol., 1 (1979) 295-319.

48 Salas, M., Guzman-Flores, C. and Schapiro, S., An ontoge- netic study of olfactory bulb electrical activity in the rat,

53

Physiol. Behav., 4 (1969) 699-703. 49 Schwob, J. E. and Price, J. L., The cortical projections of

the olfactory bulb: development in fetal and neonatal rats correlated with quantitative variation in adults, Brain Res., 151 (1978) 369-374.

50 Shepherd, G. M., Synaptic organization of the mammalian olfactory bulb, Physiol. Rev., 52 (1972) 864-917.

51 Singh, D. N. P. and Nathaniel, E. J. H., Postnatal devel- opment of mitral cell perikaryon in the olfactory bulb of the rat. A light and ultrastructural study, Anat. Rec., 189 (1977) 413-432.

52 Skeen, J. T. and Thiessen, D. D., Scent of gerbil cuisine, Physiol. Behav., 19 (1977) 11-14.

53 Taylor, P. M., Oxygen consumption in newborn rats, J. Physiol. (Lond.), 154 (1960) 153-168.

54 Teicher, M. H. and Blass, E. M., First suckling response of the newborn albino rat: the role of olfaction and amniotic fluid, Science, 198 (1977) 635-636.

55 Teicher, M. H., Stewart, W. B., Kauer, J. S. and Shepherd, G. M., Suckling pheromone stimulation of a modified glo- merular region in the developing rat olfactory bulb re- vealed by the 2-deoxyglucose method, Brain Res., (1980) 530--535.

56 Thiessen, D. and Yahr, P., The Gerbil in Behavioral Inves- tigations, Univ. Texas Press, Austin. TX, 1977.

57 Westrum, L. E., Electron microscopy of synaptic structures in olfactory cortex of early postnatal rats, J. Neurocytol., 4 (1975) 713--732.

58 Yahr, P. and Anderson-Mitchell, K., Attraction of gerbil pups to maternal nest odors: duration, specificity and ovari- an control, Physiol. Behav., 31 (1983) 241-247.

59 Yanai, .1. and Rosselli-Austin, L., Normal homing behavior in infant rats despite extensive olfactory bulb granule cell losses, Behav. Biol., 24 (1978) 539-544.