Olfactory deprivation increases dopamine D2 receptor density in the rat olfactory bulb.

Unilateral olfactory deprivation during postnatal development results in significant anatomical and neurochemical changes in the deprived olfactory bulb. Perhaps the most dramatic neurochemical change is the loss of dopaminergic expression by neurons of the glomerular region. We describe here the effects of early olfactory deprivation on other elements of the bulb dopaminergic system, namely the dopamine receptors of the olfactory bulb. Rat pups had a single naris occluded on postnatal day 2 (PN2). On PN20 or PN60, animals were sacrificed and the bulbs were examined for catecholamine levels or D2 and D1 dopamine receptor binding. Receptor densities were quantified by in vitro autoradiography using the tritiated antagonists spiperone (D2) and SCH23390 (D1). Dopamine uptake sites were similarly examined using tritiated mazindol. No significant specific labeling of D1 or mazindol sites was observed in the olfactory bulbs of control or experimental animals at either age. Normal animals displayed prominent labeling of D2 sites in the glomerular and nerve layers. After 60 days of deprivation, deprived bulbs exhibited an average increase in D2 receptor density of 32%. As determined by Scatchard analysis, the mean values for Kd and Bmax were 0.134 nM and 293 fmol/mg protein in normal bulbs, and 0.136 nM and 403 fmol/mg protein in deprived bulbs. The results suggest that, as in the neostriatum, dopamine depletion in the olfactory bulb leads to an upregulation of D2 receptor sites. This change may represent an attempt by the system to adapt neurochemically to reduced dopaminergic activity and thereby maintain bulb function.     

Single-unit analysis of postnatal olfactory learning: modified olfactory bulb output response patterns to learned attractive odors

Neonatal rats learn to approach odors associated with stimulation normally provided by their mother. The present report describes changes in olfactory bulb single-unit activity following olfactory learning in young rats. Rat pups were exposed from postnatal day 1 to 18 to either (1) peppermint-scented air while receiving tactile stimulation (Pepp-Stroked), (2) peppermint-scented air with no tactile stimulation (Pepp-Only), (3) clean air and tactile stimulation (Stroked-Only), or (4) clean air and no tactile stimulation (Naive). On day 19, single-unit activity was recorded from mitral/tufted cells in urethane-anesthetized, freely breathing pups in response to either peppermint or a novel orange odor. Mitral/tufted cell response patterns to peppermint were significantly altered in Pepp-Stroked animals compared to control pups. Peppermint exposure alone, not associated with tactile stimulation (Pepp-Only), did not affect subsequent single-cell response patterns to that odor. In addition, the modification of response patterns was specific to peppermint and was not associated with a change in respiration rate. Furthermore, Pepp-Stroked pups had a relative behavioral preference for peppermint on day 19 compared to control pups. These results demonstrate that postnatal olfactory learning selectively modifies the subsequent response patterns of olfactory bulb output cells to the attractive odor. Furthermore, these results indicate that the initial coding of an odor's attractive value occurs within the olfactory bulb.     

Unilateral odor deprivation: Time course of changes in laminar volume

Unilateral olfactory deprivation on Day 1 results in large reduction in olfactory bulb size when animals are examined in the juvenile period. The present study explored 2 issues. First, the development of changes in laminar volume was measured in order to determine when differences in olfactory bulb size emerge. Rat pups underwent surgical closure of one naris on Day 1 and laminar sizes were examined on either day 4, 8, or 12. Reliable differences emerged between "deprived" and control bulbs by Day 12. Second, to determine the length of the period of susceptibility to changes in sensory input, pups were deprived on either Day 10 or 20 and reared for thirty days. Deprivation at both ages reduced bulb size, although treatment at the later age resulted in less severe alterations.     

125I-insulin binding is decreased in olfactory bulbs of aged rats

125I-insulin binding was studied in membrane preparations of olfactory bulb, frontal cortex, hippocampus and hypothalamus from mature (5-month-old) and aged (22-month-old) rats. In the young animals the highest level of specific insulin binding was found in the olfactory bulb, with lower values of specific insulin binding in the frontal cortex, hippocampus and hypothalamus. In the aged rats the specific insulin binding was not changed in the frontal cortex, hippocampus and hypothalamus as compared to the young ones. A significant decrease of total insulin binding was observed only in the olfactory bulbs of aged rats (0.67 +/- 0.04 pmol insulin/mg protein) as compared to the mature animals (1.3 +/- 0.08 pmol insulin/mg protein). Scatchard analysis of insulin binding data revealed that this decrease was due to changes in the number of binding sites rather than to changes in the affinity of insulin receptors. It was suggested that the decrease observed in insulin receptor number in olfactory bulbs of aged rats might be due to the atrophic changes in the structure of olfactory bulbs previously shown by electron microscopy for aged rats.     

Olfactory experience modulates apoptosis in the developing olfactory bulb

Early sensory stimulation plays a key role in shaping the structure and function of the developing olfactory system. Here, we provide the first direct evidence for apoptotic cell death in the olfactory bulbs of rat pups during normal development and we also demonstrate that olfactory deprivation by unilateral naris occlusion causes a dramatic increase in apoptotic cell death in the glomerular and granule cell layers of the deprived bulb. The accessory olfactory bulbs displayed a remarkably high basal level of apoptosis but the occluded accessory bulb did not differ in that regard from the control accessory bulb. These results suggest that apoptosis may be an important mechanism by which the olfactory system can adjust its cell numbers in response to sensory stimuli experienced in early life, thereby underlying one form of plasticity in the developing olfactory system.     

Grueneberg ganglion neurons respond to cool ambient temperatures

The Grueneberg ganglion (GG) - a neuronal cell cluster of unknown function localized to the vestibule of the anterior nasal cavity - is considered as a chemosensory compartment based on the expression of olfactory receptors and the olfactory marker protein. Axonal projection of GG neurons to so-called 'necklace glomeruli' in the olfactory bulb of the brain, which are thought to be important for suckling behaviour in rodent pups, has led to the hypothesis that the GG might be involved in mother/child interactions. To survey potential activation of GG neurons in living animals during the course of mother/child interactions, expression of the activity-dependent gene c-Fos in the GG of neonatal mouse pups was monitored in the presence and absence of the dam. It was found that GG neurons were only activated in the absence of the mother. Moreover, GG activation was independent from olfactory cues as revealed by naris occlusion. Searching for stimuli eliciting GG activity in pups separated from the dam, cool ambient temperatures were found to induce strong c-Fos expression in GG neurons whereas warmer temperatures did not. These coolness-induced responses were only observed in a distinct subset of GG neurons characterized by the expression of the olfactory receptor V2r83. Finally, GG responsiveness to coolness was remarkably reduced in older stages. In summary, these findings suggest that the GG of neonatal mice is activated by cool ambient temperatures to which they are exposed in the absence of their dam, indicating that the GG might function as a thermosensor.     

Early unilateral deprivation modifies olfactory bulb function

Unilateral olfactory deprivation during postnatal development produces significant structural and neurochemical modifications of the olfactory bulb. In the present report, we describe the functional consequences of such deprivation. Rat pups had a single naris occluded on postnatal day 2 (PN2) to deprive them of early olfactory stimulation. On PN20-22, the occluded naris was reopened, the previously open naris was sealed, and responses of the deprived olfactory bulb to odors were assessed using both single-unit recording from mitral/tufted cells and quantitative 14C-2-deoxyglucose (2-DG) autoradiography. While the response properties of individual odor-stimulated mitral/tufted cells were not altered by early deprivation, spontaneous activity was depressed, and there was a significantly higher incidence of odor-responsive mitral/tufted cells in deprived compared to nondeprived bulbs. In addition, odor-stimulated deprived bulbs demonstrated greater uptake of 2-DG than did non-deprived bulbs. Together, these data indicate that the olfactory system demonstrates an increased responsiveness to sensory cues following early deprivation.     

The effect of intranasal zinc sulfate treatment on odor-mediated behavior and on odor-induced metabolic activity in the olfactory bulbs of neonatal rats

This study describes the effect of lesions of the peripheral olfactory receptor sheet on odor-mediated behavior and on odor-induced metabolic activity in the neonatal olfactory bulb. Nine-day-old rats were treated by intranasal irrigation with a solution of 1% ZnSO4 or 5% ZnSO4 or saline. At 1 and 5 days following treatment they were tested for maternally-directed behaviors which are mediated in large part by olfactory cues. At 1 day following treatment the pups treated with ZnSO4 solutions had significant deficits in their odor-directed behavior. By 5 days however, most pups treated with 1% ZnSO4 had recovered to control levels, while the pups treated with 5% ZnSO4 continued to exhibit profound behavioral deficits. Following the behavioral testing, the pups were tested using the 2-deoxyglucose method for amyl acetate odor-induced metabolic activity in their olfactory bulbs. Control pups had large regions of high focal activity in their anterolateral and caudomedial olfactory bulbs. The 1% ZnSO4 pups had smaller foci which were confined primarily to the medial olfactory bulb. The 5% ZnSO4 pups had very little focal uptake in their olfactory bulbs. There was a clear correlation between intact olfactory nerve and glomerular layers and sites of increased functional activity. The results of this study demonstrate that there is a relationship between the degree of behavioral deficit and the extent of odor-induced functional activity in the olfactory bulb.     

Modulation of olfactory learning in young rats through intrabulbar GABA(B) receptors

After training with an odour paired with foot shock on postnatal day 11, rat pups show an aversion to the odour in testing on postnatal day 12. The mechanisms underlying this aversive olfactory learning involve disinhibition of mitral/tufted cells in the olfactory bulb by the somatosensory stimulation-induced activation of centrifugal noradrenergic fibres originating in the locus coeruleus. The activity of mitral/tufted cells is regulated through gamma-aminobutyric acidA (GABA(A)) receptors in the external plexiform layer and GABA(B) receptors in the glomerular layer. We have previously presented that aversive olfactory learning in young rats is modulated through GABA(A) receptors in the olfactory bulb. In the present study we examined the consequence of manipulating GABA(B) receptors in the olfactory bulb during training. Baclofen, a GABA(B) receptor agonist when infused into the olfactory bulb during the pairing of an odour with foot shock, prevented aversive olfactory learning in a dose-dependent manner. Infusion of saclofen, a GABA(B) receptor antagonist, during training with a citral odour in the absence of foot shock produced aversive responses not only to the odour, but also to strange odours (benzaldehyde and vanillin) not previously presented. Such olfactory aversions were observed even if saclofen was infused without odour exposure. These results suggest that olfactory learning in young rats is modulated through GABA(B) receptors in the olfactory bulb.     

Regional autonomy in the peripheral processing of odor signals in newborn rabbits

In newborn rabbits, small and apparently arbitrary regions of the olfactory bulb and associated epithelium appear capable of a high degree of odor processing. After medial or lateral removal of up to 80% of the olfactory bulbs, including the accessory bulb, newborn pups were still able to respond appropriately to the pheromone-governing suckling behavior (Expt. I), could rapidly learn to associate a novel, artificial odor with suckling (Expt. II), and continued to respond appropriately to artificial odors learned prior to lesioning (Expt. III). These findings suggest that the perception and recognition of such suckling signals does not depend on the integration of information from the entire bulb or epithelium, and question the extent to which patterns of 2-deoxyglucose uptake in the bulb reflect the neural coding for specific odors. However, as the tasks set here only required detection of odor signals and not true odor discrimination, it may still be that the full bulbar pattern of activation is necessary for higher-order processing, such as distinguishing between odors and attributing different meanings to them.     

Combined modulating effects of the general arousal and the specific hunger arousal on the olfactory bulb responses in the rat.

The combined modulating effects of the general level of arousal and specific hunger arousal on the olfactory bulb responses were investigated in the rat. Vigilance state parameters and multi-unit mitral cell activity were recorded in freely moving animals stimulated by control odours and by their usual food odour, either in the hungry or the satiated state. The nutritionally modulated bulb responses towards food odour were observed only for high arousal level (wakefulness). In rapid eye movement sleep (REMS), no olfactory response occurred. In slow wave sleep (SWS), one observed either a high bulb responsiveness to stimuli with neocortical arousal, or a general inexcitability. Each odorous stimulus in SWS elicited a higher neocortical arousal rate in the hungry than in the satiated state, as did food odour compared with control odours in both nutritional states. In SWS, a progressive alteration of the nutritionally modulated responses occurred at first at the bulb level and later for inner structures. Rats fed 2 h a day displayed a reversed circadian sleep-waking cycle and a lower SWS proportion compared with rats fed ad libitum. The hunger arousal could quantitatively and qualitatively modulate the activity of structures regulating the sleep-waking pattern.     

The role of olfactory bulb norepinephrine in early olfactory learning.

Wistar rat pups were implanted with bilateral olfactory bulb cannulas on postnatal day 5 (PN5). On PN6, pups were trained in an olfactory classical conditioning task with peppermint odor as the CS and tactile stimulation/stroking as the UCS. Pups were randomly assigned to either PAIRED, BACKWARD or ODOR-only conditions. Half the pups in each group received intrabulbar infusions of 100 microM propranolol and half received intrabulbar infusions of saline during the training session. Propranolol infusions blocked acquisition of the learned odor preference expressed by PAIRED saline-infused pups. Diffusion of the infusate was checked in additional pups by infusing [3H]NE and performing LSC analysis. Infusate concentration did not significantly differ between the anterior and posterior halves of the bulb, but were sharply lower in the olfactory peduncle and more posterior areas. The results suggest that olfactory bulb NE is critical for early olfactory learning.     

NMDA receptor activation and early olfactory learning

Norway rat pups have an enhanced olfactory bulb response to odors which they have learned to prefer early in life. When N-methyl-D-aspartate receptors are blocked pharmacologically before olfactory preference training, both the behavioral preference and the enhanced olfactory bulb response to the learned odor are suppressed. These results implicate the activation of these receptors in the kind of neural and behavioral plasticity that normally occurs during development.     

Localization of Nitric Oxide Synthase in the Mouse Olfactory and Vomeronasal System: a Histochemical, Immunological and In Situ Hybridization Study

The distribution of nitric oxide synthase (NOS) in the mouse olfactory bulb and olfactory epithelium, including the vomeronasal organ, was studied using an anti-NOS antibody, NADPH diaphorase histochemistry and in situ hybridization with NOS specific antisense oligonucleotide probes. Interneurons containing NOS protein and mRNA, and exhibiting NADPH diaphorase activity were detected in the plexiform layer of the main olfactory bulb and the granule cell layer of main and accessory olfactory bulbs. Periglomerular cells and granule cells in the main olfactory bulb were also NOS positive with diaphorase and immunostaining for NOS. In contrast, no evidence for NOS expression was found either in the main olfactory epithelium or in the vomeronasal organ, in spite of the strong diaphorase staining of the surface of the main olfactory epithelium. Polymerase chain reaction amplification experiments for detection of NOS gene expression further indicated that NOS is expressed in the olfactory bulb but not in either the main olfactory epithelium or vomeronasal organ. Use of an antibody raised against another enzyme, NADPH-P450 oxidoreductase, showed that this protein was strongly expressed in the olfactory epithelium. Activity of this enzyme may account for the diaphorase histochemical staining of the epithelia. An involvement of neuronal nitric oxide synthase in signalling in olfactory receptor neurons is therefore doubtful, although NOS is clearly expressed in neurons in both main and accessory olfactory bulbs.     

Focal 2-DG uptake persists following olfactory bulb lesions

Exposure of rats to different odors produces spatially distinct patterns of ¹⁴C-2-deoxyglucoss uptake (2-DG) in the glomerular layer of the main olfactory bulb. However, lesions of specific regions of the bulb that reliably contain 2-DG foci reportedly do not impair the ability of rats to perform olfactory-guided behaviors, suggesting that the lesioned olfactory bulb retains odor-responsiveness. Because the absence of focal 2-DG incorporation in lesioned olfactory bulbs has not been verified by 2-DG autoradiography, it cannot be concluded that focal responses in the olfactory bulb do not contribute to the encoding of olfactory information. To examine the effects of bulb lesions on 2-DG uptake in the olfactory bulb, we placed lesions in specific regions of the bulb that reliably contain 2-DG foci. We then exposed rats to odors 3 or 6 weeks later to determine if the lesions effectively eliminated focal 2-DG uptake in these bulbs. The results indicate that lesioned olfactory bulbs contain focal regions of 2-DG uptake in response to odor stimulation.     

Regulation of c-Fos mRNA and fos protein expression in olfactory bulbs from unilaterally odor-deprived adult mice

Odorant deprivation, produced by unilateral naris closure, profoundly reduces tyrosine hydroxylase (TH) expression within intrinsic olfactory bulb dopamine neurons. The TH gene contains an AP-1 site, which interacts with the product of the immediate early gene, c-fos. c-Fos exhibits activity dependent regulation in the CNS. The hypothesis that odorant stimulation and deprivation might modify c-fos expression in TH neurons was tested in adult CD-1 mice, subjected to unilateral naris closure. After 2 months, naris closed and control mice were exposed to either clean air for 60 min or clean air for 60 min followed by 30 min of alternating exposure to 10% isoamyl acetate (1 min) and air (4 min). A parallel reduction occurred in TH and fos expression (both c-fos mRNA and fos-like immunoreactivity) in the glomerular layer of the odorant-deprived olfactory bulb. Odor stimulation induced a short-lived increase in c-fos mRNA and fos-like immunoreactivity in olfactory bulbs contralateral to naris closure. The increase in fos expression was region-specific in the glomerular layer but more diffuse in mitral and granule cell layers. In olfactory bulbs ipsilateral to naris closure, odor stimulation also induced c-fos mRNA expression in the mitral and granule cell layers and sparsely within limited periglomerular regions. Odor induced expression in mitral and granule cell layers may represent increased centrifugal activity acting on as yet unknown genes. These results suggest a correlation between c-fos mRNA expression and increased neuronal activity in the olfactory bulb which, in turn, acts to regulate TH expression in periglomerular neurons.     

The locus coeruleus, norepinephrine, and memory in newborns

Use of learned odor cues by newborn rats is critical for pup survival. Rat pups acquire approach responses to maternal odors through an associative conditioning mechanism. This learned behavioral response is accompanied by a modification of olfactory bulb neural response patterns to the learned odor. Both the behavioral and neural reponse changes involved and require norepinephrine release in the olfactory bulb. The source of this norepinephrine is the locus coeruleus. It is proposed that the unique response properties of the locus coeruleus during the early postnatal period in the rat may facilitate acquisition of these critical early memories.     

Deafferentation-induced changes in the olfactory bulb of adult zebrafish

The influence of the olfactory organ on maintenance of olfactory bulb structure was examined in zebrafish, using peripheral deafferentation. This fish provides a model in which the olfactory organ is easily accessible for removal, the animals easily survive the surgery, and the olfactory bulbs are small enough to allow rigorous analysis of the resulting effects. Unilateral olfactory organ ablations were performed on anesthetized adult zebrafish using a small-vessel cautery iron. Fish were allowed to survive for 1, 3, or 6 weeks following the procedure. Analysis of deafferented animals revealed that most, if not all, of the olfactory organ was missing on the ablated side, and the structure did not regenerate. The morphology of the olfactory bulb was affected notably by the removal of its primary afferent innervation. The olfactory nerve layer was diminished at 1 week and absent by 3 weeks post-deafferentation. At all of the survival times the deafferented bulb appeared significantly smaller at the gross level, and there was a statistically significant effect on bulb size and cell number after 6 weeks. Tyrosine hydroxylase expression, as revealed by immunohistochemistry, was decreased noticeably on the ablated side. In conclusion, the olfactory organ is important in the preservation of normal olfactory bulb anatomy and neurochemistry in adult zebrafish. Thus, the influence of the periphery does not end with the formation of the mature olfactory bulb.     

Cerebral lateralization of olfactory-mediated affective processes in rats

To determine whether processing of information is lateralized in the brain of non-human mammalian species, rats that had undergone ablation of the left or right olfactory bulb were compared to sham-operated animals and to bilaterally bulbectomized animals in their response to emotionally positive or negative social odours. Left-bulbectomized rats were impaired in their behavioural reaction but not in their hormonal response to an odour from a stressed conspecific. They fully retained, however, their ability to recognize a nonstressed juvenile conspecific on the basis of its olfactory characteristics. These results suggest that hemispheric asymmetries develop in mammals not for recognition of emotional stimuli but for association of emotional experiences with appropriate adaptive behaviour.     

Somatostatin receptors increase in the olfactory bulb of developing pups after perinatal exposure to cocaine

The effects of chronic prenatal and/or postnatal exposure to cocaine on somatostatin concentration and receptors were studied in the olfactory bulbs of rat pups at birth and at 15 days old. Wistar rats were injected subcutaneously with single daily doses of 40 mg cocaine hydrochloride/kg from days 7 to 19 of gestation, from day 7 of gestation to day 15 postpartum or from parturation to day 15 postpartum. Fetal exposure to cocaine decreased SS concentrations in the olfactory bulb of the newborn pups while prenatal-plus-postnatal exposure increased this parameter. Administration of cocaine only during lactation did not induce any change. Exposure during gestation or during nursing induced an increase in the total number of somatostatin receptors and a decrease in the affinity constant in the olfactory bulb of newborn and 15-day-old pups. These results suggest that the development of somatostatin receptors in the olfactory bulb can be altered by prenatal and/or nursing period exposure to cocaine.