Dissociation of behavioral and neural correlates of early associative learning

Wistar rat pups were trained in an olfactory associative conditioning task on postnatal Day 6, 12, or 20. The training consisted of 20 pairings of a novel odor (peppermint) with footshock (1.5 mA, 1 s) with an intertrial interval of 3 min. Additional pups were trained in either unpaired or naive control conditions. On the day following training, pups were either tested for their behavioral response to the conditioned odor in a two-odor choice test, or injected with ¹⁴C-2-deoxyglucose and exposed to the odor for examination of olfactory bulb neural responses to the odor. The results demonstrate that, although pups at all ages learned to avoid the odor, only pups trained during the first postnatal week had a modified olfactory-bulb glomerular-layer response to the odor. These results suggest that although olfactory memory is correlated with modification of olfactory bulb glomerular layer function in newborns, these changes are not required for normal memory in older pups. © 1995 John Wiley & Sons, Inc.     

Gabaergic control of olfactory learning in young rats.

Olfactory learning in young rats correlates with neural plasticity in the olfactory bulb, and involves noradrenergic modulation of reciprocal dendrodendritic synapses between mitral cells and GABAergic granule cells. The purpose of this study was to examine, in vivo, the consequences of manipulating bulbar GABA transmission during training. In the first experiment, postnatal day 11 rat pups were trained in an olfactory associative learning task with citral odor and foot shock as the conditioned and unconditioned stimuli, respectively. The pups received continuous infusion of saline or the GABA(A) receptor agonist muscimol into the olfactory bulbs throughout a 30-min training session. The pups were then tested on postnatal day 12 for a preference for or an aversion to citral odor. Saline-infused control pups developed an aversion to citral odor. The GABA(A) receptor agonist muscimol impaired this aversive learning in a dose-dependent manner. In the second experiment, pups were exposed to the odor for 30 min while receiving continuous intrabulbar infusion of a low or high dose of the GABA(A) receptor antagonist bicuculline, without any other reinforcer. Depending on whether a low (0.2 nmol/bulb) or high (1.0 nmol/bulb) dose of bicuculline was infused, the pups showed a preference or an aversion for citral odor after infusion of low and high doses, respectively. These results indicate that disinhibition of mitral cells in the olfactory bulb is critical for olfactory learning in young rats, and suggest that the degree of disinhibition is an important determinant in acquiring either preference or aversion for the conditioned odor.     

Developmental change in the access to olfactory memories

Memory for a learned odor preference can be functionally confined to one side of the brain in 6-day-old rat pups by preferentially stimulating a single naris and corresponding olfactory bulb during training. We report here that this form of unilateral learning is present only during the first postnatal week; older pups show bilateral recall of unilateral olfactory experience. The maturation of bilateral learning probably depends on the postnatal growth and development of olfactory commissural fibers, because infantlike unilateral learning and memory is reinstated when these commissural fibers are sectioned before training in older pups. Section of commissural fibers after training also resulted in unilateral preferences. This latter finding indicates that the learned odor preference of older pups tested with the untrained naris open depends on access to unilaterally stored memories on the contralateral side, access provided by the newly developed commissural projections.     

Developmental change in unilateral olfactory habituation is mediated by anterior commissure maturation

Habituation of an orienting response in rat pups was used to study the development of the anterior commissure (AC) and its role in olfactory memory. Six- and 12-day-old pups received odor presentations to one side of their olfactory system and were tested later for habituation to additional presentations made to either the trained or untrained side. Six-day-old pups remembered only on the trained side. Twelve-day-old pups remembered when tested on either side. Transection of the AC in 12-day-old pups before, but not after, training prevented the transfer of memory. Thus, between 6 and 12 days of age, olfactory cross-projections carried in the AC mature and provide a functional substrate for transfer, at the time of training, of a form of olfactory learning.     

Association of an odor with activation of olfactory bulb noradrenergic beta-receptors or locus coeruleus stimulation is sufficient to produce learned approach responses to that odor in neonatal rats

These experiments examined the sufficiency of pairing an odor with either intrabulbar activation of noradrenergic beta-receptors or pharmacological stimulation of the locus coeruleus to support learned odor preferences in Postnatal Day 6-7 rat pups. The results showed that pups exposed to odor paired with beta-receptor activation limited to the olfactory bulb (isoproterenol, 50 microM) displayed a conditioned approach response on subsequent exposure to that odor. Furthermore, putative stimulation of the locus coeruleus (2 microM idazoxan or 2 mM acetylcholine) paired with odor produced a subsequent preference for that odor. The effects of locus coeruleus stimulation could be blocked by a pretraining injection of the beta-receptor antagonist propranolol (20 mg/kg). Together these results suggest that convergence of odor input with norepinephrine release from the locus coeruleus terminals within the olfactory bulb is sufficient to support olfactory learning.     

Behavioral and neural correlates of postnatal olfactory conditioning: I. Effect of respiration on conditioned neural responses

Following olfactory classical conditioning, infant rats exhibit a preference for the conditioned odor and exhibit enhanced uptake of focal 14C 2-deoxyglucose (2-DG) within the olfactory bulb. The present experiments assessed the role of respiration on the expression of the enhanced 2-DG uptake response. Pups were conditioned from postnatal day (PN) 1-18 with an olfactory stimulus paired with a reinforcing tactile stimulus which mimics maternal contact (Odor-Stroke). Control pups received odor only or tactile stimulation only. On PN 19, pups received 1 of 3 tests: 1) a two-odor choice test, 2) an odor/2-DG test with normal respiration allowed, or 3) an odor/2-DG test with respiration experimentally controlled. The results indicated that: 1) Odor-Stroke pups learned the conditioned odor preference, 2) Odor-Stroke, normally respiring pups exhibited enhanced olfactory bulb 2-DG uptake when compared to control pups. No difference in respiration rate was detected between groups in normally respiring pups. 3) Odor Stroke pups whose breathing was experimentally controlled exhibited enhanced olfactory bulb 2-DG uptake when compared to control pups with an identical number of respirations. Together, these results demonstrate that modified respiration during testing is not required for the expression of a modified olfactory bulb response to learned attractive odors. Therefore, the data suggest that the olfactory system itself is modified by early learning.     

Calcineurin inhibition eliminates the normal inverted U curve,enhances acquisition and prolongs memory in a mammalian 3′-5′-cyclic AMP–dependent learning paradigm

The role protein phosphatase 2B (calcineurin, CaN) plays in learning and memory has received a significant amount of attention due to its promotion of the dephosphorylation of 3′-5′-cyclic AMP response element binding protein (CREB). Researchers have ascertained that overexpression of CaN is associated with memory retention deficits [Foster TC, Sharrow KM, Masse JR, Norris CM, Kumar A (2001) Calcineurin links Ca²⁺ dysregulation with brain aging. J Neurosci 21:4066–4073; Mansuy IM, Mayford M, Jacob B, Kandel ER, Bach ME (1998) Restricted and regulated overexpression reveals calcineurin as a key component in the transition from short-term to long-term memory. Cell 92:39–49], while CaN inhibition enhances learning and memory [Gerdjikov TV, Beninger RJ (2005) Differential effects of calcineurin inhibition and protein kinase A activation on nucleus accumbens amphetamine-produced conditioned place preference in rats. Eur J Neurosci 22:697–705; Ikegami S, Inokuchi K (2000) Antisense DNA against calcineurin facilitates memory in contextual fear conditioning by lowering the threshold for hippocampal long-term potentiation induction. Neuroscience 98:637–646]. The present study hypothesized that infusion of a CaN inhibitor (FK506) bilaterally into the olfactory bulbs of postnatal day 6 Sprague Dawley rat pups would prolong the duration of a conditioned odor preference and retard cyclic AMP response element binding protein dephosphorylation. A 2 mg/kg s.c. injection of isoproterenol (ISO, β-adrenoceptor agonist) was paired with a 10 min exposure to peppermint and subsequently an infusion of FK506. Immunohistochemistry for phosphorylated 3′-5′-cyclic AMP response element binding protein (pCREB) revealed that unilateral infusion of FK506 resulted in an amplification of phosphorylated CREB in the olfactory bulb 40 min after training compared with saline-infused bulbs. Pups infused bilaterally with FK506 maintained a learned preference for peppermint 48, 72 and 96 h after training. CaN inhibition also modified the conventional inverted U curve obtained when ISO is used to replace stroking, as the unconditioned stimulus. When pups were infused with FK506, learning occurred with sub- and supra-optimal doses of ISO indicating that CaN overcomes non-optimal effects ISO may have on learning. We demonstrate that CaN inhibition can extend the duration of conditioned olfactory memory and may provide a target for memory prolongation that is superior to even phosphodiesterase inhibition observed in previous studies.     

Facilitatory effect of ritanserin is mediated by dopamine D(1) receptors on olfactory learning in young rats

The olfactory bulb is critically involved in early olfactory learning. In this study, we examined the effect of intrabulbar infusion of ritanserin, a 5-hydroxytryptamine(2) (5-HT(2)) receptor antagonist on a one-trial aversive olfactory learning in young rats. Ritanserin, a 5-HT(2) receptor antagonist, was continuously infused into the olfactory bulb of postnatal day-11 (PND 11) rat pups during a 30-min training session of pairing citral odor and foot shock. On the following day, the time spent in the part of the apparatus where the odor was present was measured as an index of odor aversion. Consistent with a previous study on olfactory preference learning, 1 nM ritanserin, but not 10 nM, blocked the olfactory aversive learning. We further examined the ability of 10 nM ritanserin to induce olfactory learning in the absence of the unconditioned stimulus foot shock. Pups that received intrabulbar infusion of 10 nM ritanserin in the presence of citral odor developed an aversion to the odor without foot shock. Since ritanserin has been shown to have an affinity for dopamine receptors, we examined the effect of dopamine antagonists on the ritanserin-induced aversive olfactory learning. Co-infusion of the dopamine D(1) receptor antagonist (+/-)-SKF-83566 with ritanserin dose-dependently prevented induced learning. In contrast, the D(2) receptor antagonist spiperone was without effect. These results extend the previous finding on the role of bulbar 5-HT(2) receptors in early olfactory learning and suggest that high concentration of ritanserin facilitates aversive olfactory learning through D(1) receptors in the olfactory bulb.     

Consolidation and expression of a shock-induced odor preference in rat pups is facilitated by opioids

To support nipple attachment and huddling, rat pups must learn to approach and prefer maternal odor. Similar to other altricial species, rat pups have a sensitive period for learning this odor preference, which ends around postnatal day (PN) 10 and coincides with the emergence of walking. One characteristic of this sensitive period is that an odor paired with moderate shock elicits an odor preference. After PN10, this behavioral training produces an odor aversion, although pain threshold remains unchanged. Recently, we demonstrated that the endogenous opioid system might be a key element in the acquisition of the shock-induced odor preference during the sensitive period since antagonism of this system disrupts odor preference learning. In older pups, acquisition of a shock-induced odor aversion was unaffected by opioid system manipulation. The purpose of these experiments was to further elucidate the role of opioids in infant olfactory learning through assessment of memory consolidation and expression during and after the sensitive period. In Experiment 1, we demonstrate that naltrexone (NTX), a nonspecific opioid antagonist, given immediately following odor-shock conditioning during the sensitive period, blocks odor preference formation and yields an odor aversion. However, the same treatment does not disrupt consolidation of an odor aversion in older pups. In Experiment 2, we demonstrate that during the sensitive period, NTX disrupts expression of the shock-induced odor preference, but not the learned odor aversion in older pups. Results using this model of attachment suggest that opioids have an important role in the acquisition, consolidation, and expression of early olfactory preferences. Furthermore, since prenatal drug exposure is known to alter the endogenous opioid system, these results highlight the capacity of prenatal opiate exposure to disrupt early infant learning and attachment.     

Corticosterone influences on Mammalian neonatal sensitive-period learning

Infant rats exhibit sensitive-period odor learning characterized by olfactory bulb neural changes and odor preference acquisitions critical for survival. This sensitive period is coincident with low endogenous corticosterone (CORT) levels and stress hyporesponsivity. The authors hypothesized that low corticosterone levels modulate sensitive-period learning. They assessed the effects of manipulating CORT levels by increasing and removing CORT during (Postnatal Day 8) and after (Postnatal Day 12) the sensitive period. Results show that (a) exogenous CORT prematurely ends sensitive-period odor-shock-induced preferences; (b) adrenalectomy developmentally extends the sensitive period as indicated by odor-shock-induced odor-preference learning in older pups, whereas CORT replacement can reinstate fear learning; and (c) CORT manipulation modulates olfactory bulb correlates of sensitive-period odor learning in a manner consistent with behavior.     

Neural correlates of conditioned odor avoidance in infant rats

Newborn rat pups can learn to either approach or avoid odor cues through associative conditioning. The present results demonstrate that preference conditioning and avoidance conditioning both modify olfactory bulb responses (focal 2-deoxyglucose uptake and mitral-tufted cell single unit responses) to the conditioned odor. Despite opposing behavioral responses to the conditioned odor, however, olfactory bulb neural responses did not detectably differ between learned odor cues signaling approach and those signaling avoidance. Control pups exhibited neither the behavioral nor neural changes. Furthermore, both the behavioral and neural changes to these odor cues could be extinguished. These results suggest that the olfactory bulb in neonates may code learned odor importance, but specific information attached to that importance may require processing in other brain regions.     

Spatial coding of enantiomers in the rat olfactory bulb

Because of their unique properties, enantiomers (pairs of mirror-symmetric, nonsuperimposable molecules that differ only in optical activity and their interaction with other chiral molecules) have been instrumental in demonstrating that olfactory perception relies on molecular shape. To investigate how molecular structure is encoded by the olfactory system, we combined behavioral discrimination tasks with optical imaging of intrinsic signals. We found that rats can behaviorally discriminate members of a wide range of enantiomer pairs, and imaging revealed enantiomer-selective glomeruli in the olfactory bulb, indicating that the spatial pattern of glomerular activity provides sufficient information to discriminate molecular shape.     

Activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase signaling pathway leading to cyclic AMP response element-binding protein phosphorylation is required for the long-term facilitation process of aversive olfactory learning in young rats

The mitogen-activated protein kinase/extracellular-signal regulated kinase (MAPK/ERK) cascade is an important contributor to synaptic plasticity that underlies learning and memory. ERK activation by the MAPK/ERK kinase (MEK) leading to cyclic-AMP response element binding protein (CREB) phosphorylation is implicated in the formation of long-term memory. We have demonstrated that CREB phosphorylation in the olfactory bulb (OB) is important for aversive olfactory learning in young rats, yet whether MAPK/ERK functions as an upstream regulator are necessary for this olfactory learning remains to be determined. Therefore, we addressed this issue using behavioral and Western blot analyses. The MEK inhibitor PD98059 was continuously infused into the OB of postnatal day 11 rat pups during a 30-min training session regarding the pairing of citral odor and foot shock. On the following day, the time spent in the part of the apparatus where the odor was present was measured as an index of odor aversion. PD98059 impaired olfactory learning in a dose-dependent manner without affecting memory retention 1 h after training. We further tested whether odor-shock training leads to MAPK/ERK activation in the OB and defines the time course of the activation. Phosphorylated ERKs (P-ERKs) 1 and 2 were significantly increased for 60 min after the training without changes in total ERKs 1 and 2. By contrast, intrabulbar infusion of PD98059 during the training significantly reduced P-ERKs 1 and 2 as well as phosphorylated CREB without any effects on the total ERKs or CREB. Taken together with the previous findings, these results indicate that the MAPK/ERK-CREB pathway is required for the long-term, but not the short-term, facilitation process of aversive olfactory learning in young rats.     

Noradrenergic neuromodulation in the olfactory bulb modulates odor habituation and spontaneous discrimination

Noradrenergic projections from the locus coeruleus (LC) project to the olfactory bulb (OB), a cortical structure implicated in odor learning and perceptual differentiation among similar odorants. The authors tested the role of OB noradrenaline (NA) in short-term olfactory memory using an animal model of LC degeneration coupled with intrabulbar infusions of NA. Specifically, the authors lesioned cortical noradrenergic fibers in mice with the noradrenergic neurotoxin N-Ethyl-N-(2-chloroethyl)-2-bromobenzylamine hydrochloride (DSP4) and measured the effects on an olfactory habituation/spontaneous discrimination task. DSP4-treated mice failed to habituate to repeated odor presentations, indicating that they could not remember odors over the 5-min intertrial interval. The authors then infused NA bilaterally into the OBs of both DSP4-treated and nonlesioned control animals at two concentrations (10(-3)M and 10(-5)M, 2 microl/side). In DSP4-treated animals, NA administration at either concentration restored normal habituation and spontaneous discrimination performance, indicating that noradrenergic neuromodulation mediates these aspects of perceptual learning and that its efficacy does not require activity-dependent local regulation of NA release. Functional OB learning mechanisms may be necessary for normal odor recognition and differentiation among physically similar odorants.     

Endogenous opioids and their role in odor preference acquisition and consolidation following odor-shock conditioning in infant rats.

We assessed the neurochemical basis of olfactory learning induced by presentations of odor and moderate shock in infant rats. Paradoxically, shock conditioning produces an odor preference in 8-day-olds, but an odor aversion in 12-day-olds. Studies have demonstrated the importance of opioids in early olfactory learning; their specific role remains undefined. In this study, postnatal Days 8 and 12 pups were systemically injected with naltrexone, a nonspecific opioid antagonist, or saline and received either paired or backward presentations of odor-moderate shock or odor-only presentations. Blocking the opioid system during conditioning disrupted acquisition of the Day 8 odor preference, but not the Day 12 odor aversion. Additional Day 8 pups were given naltrexone posttraining. Naltrexone not only blocked consolidation of an odor preference but also yielded an odor aversion. These results suggest that the opioid system has a critical role in both olfactory learning and consolidation of odor preferences during the sensitive period.     

Spatial patterns of olfactory bulb single-unit responses to learned olfactory cues in young rats

1. Neonatal rat pups were classically conditioned to an odor stimulus from postnatal day 1 (PN1) to PN18. Tactile stimulation (stroking) was used as the unconditioned stimulus. On PN19, mitral/tufted cell single-unit responses to the conditioned odor were examined in both conditioned and control pups. Recordings were made from mitral/tufted cells in two regions of the olfactory bulb: 1) an area typically associated with focal [14C]2-deoxyglucose (2-DG) uptake in response to the conditioned odor and 2) an area distant from focal 2-DG uptake to the conditioned odor. Animals were anesthetized with urethane and were naturally respiring during the single-unit recording procedure. 2. Changes in mitral/tufted cell firing rate in response to odors in both bulbar regions and all training groups were classified as either excitatory, suppressive, or no response. This response classification was used to compare response patterns to the conditioned odor between bulbar regions and training groups. 3. Classical conditioning selectively modified the response patterns of mitral/tufted cells to the conditioned odor when those cells were associated with regions of focal 2-DG uptake for that odor. Mitral/tufted cells demonstrated significantly more suppressive and fewer excitatory responses to the conditioned odor than cells in control pups. Response patterns to a novel odor were not similarly modified. 4. Response patterns of mitral/tufted cells distant from the focal region of 2-DG uptake to the conditioned odor were not modified by conditioning compared with control pups. 5. The difference in response pattern between cells in the 2-DG focus and cells distant to the 2-DG focus was apparent within 500 ms of the stimulus onset. Given the respiratory rate of these pups (2 Hz), these data suggest that the modified response pattern occurred on the first inhalation of the learned odor. 6. These data demonstrate that both spatial and temporal patterns of olfactory bulb output neuron activity are used in the coding of olfactory information in the bulb. Furthermore, these spatial/temporal response patterns can be modified by early learning.     

Locus coeruleus degeneration exacerbates olfactory deficits in APP/PS1 transgenic mice

Neuronal loss in the locus coeruleus (LC) is 1 of the early pathological events in Alzheimer's disease (AD). Projections of noradrenergic neurons of the LC innervate the olfactory bulb (OB). Because olfactory deficits have been reported in early AD, we investigated the effect of induced LC degeneration on olfactory memory and discrimination in an AD mouse model. LC degeneration was induced by treating APP/PS1 mice with N-(2-chloroethyl)-N-ethyl-bromo-benzylamine (DSP4) repeatedly between 3 and 12 months of age. Short term odor retention, ability for spontaneous habituation to an odor, and spontaneous odor discrimination were assessed by behavioral tests. DSP4 treatment in APP/PS1 mice resulted in an exacerbation of short term olfactory memory deficits and more discrete weakening of olfactory discrimination abilities, suggesting that LC degeneration contributes to olfactory deficits observed in AD. Importantly, DSP4 treatment also increased amyloid β (Aβ) deposition in the olfactory bulb of APP/PS1 mice, which correlated with olfactory memory, not with discrimination deficits.     

Neonatal handling and the maternal odor preference in rat pups: Involvement of monoamines and cyclic AMP response element-binding protein pathway in the olfactory bulb

Early-life environmental events, such as the handling procedure, can induce long-lasting alterations upon several behavioral and neuroendocrine systems. However, the changes within the pups that could be causally related to the effects in adulthood are still poorly understood. In the present study, we analyzed the effects of neonatal handling on behavioral (maternal odor preference) and biochemical (cyclic AMP response element-binding protein (CREB) phosphorylation, noradrenaline (NA), and serotonin (5-HT) levels in the olfactory bulb (OB)) parameters in 7-day-old male and female rat pups. Repeated handling (RH) abolished preference for the maternal odor in female pups compared with nonhandled (NH) and the single-handled (SH) ones, while in RH males the preference was not different than NH and SH groups. In both male and female pups, RH decreased NA activity in the OB, but 5-HT activity increased only in males. Since preference for the maternal odor involves the synergic action of NA and 5-HT in the OB, the maintenance of the behavior in RH males could be related to the increased 5-HT activity, in spite of reduction in the NA activity in the OB. RH did not alter CREB phosphorylation in the OB of both male and females compared with NH pups. The repeated handling procedure can affect the behavior of rat pups in response to the maternal odor and biochemical parameters related to the olfactory learning mechanism. Sex differences were already detected in 7-day-old pups. Although the responsiveness of the hypothalamic–pituitary–adrenal axis to stressors is reduced in the neonatal period, environmental interventions may impact behavioral and biochemical mechanisms relevant to the animal at that early age.