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.     

Non-specific olfactory aversion induced by intrabulbar infusion of the GABA(A) receptor antagonist bicuculline in young rats

On postnatal day 12, young rats show an aversion to an odor to which they had been exposed along with presentations of foot shock on postnatal day 11. The acquisition of this aversive learning involves and requires disinhibition of the mitral/tufted cells induced by centrifugal noradrenergic activation during somatosensory stimulation. This olfactory learning is established only for the odor to which the rat has been exposed during conditioning. Infusion of the GABA(A) receptor antagonist bicuculline at a high dose (2.0 nmol/each olfactory bulb) into the olfactory bulb in the presence of an odor is capable of developing olfactory aversive responses without somatosensory stimulation in young rats. The purpose of this study is to characterize the properties of bicuculline-induced aversive responses. In contrast to the odor specificity of aversive learning produced by odor-shock conditioning, bicuculline-induced aversive responses lack odor specificity. Namely, bicuculline infusion in the presence of a citral odor results, in a dose-dependent manner, in subsequent aversive responses to strange odors (benzaldehyde and vanillin) that have never been presented. Moreover, bicuculline infusion alone is sufficient to produce dose-dependent aversive responses to strange odors (citral, benzaldehyde and geraniol).From these results we suggest that disinhibition of mitral/tufted cells from granule cells by bicuculline infusion makes young rats aversive to strange odors non-specifically, as if the rats had learned the odor aversion as a result of odor exposure paired with foot shock. Different mechanisms of disinhibition of the mitral/tufted cells may underlie both the pharmacological manipulation and noradrenergic activation by somatosensory stimulation.     

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.     

Common properties between synaptic plasticity in the main olfactory bulb and olfactory learning in young rats

Aversive olfactory learning was established in young rats after odor exposure paired with foot shock through a classical conditioning paradigm. Using behavioral pharmacology and Western blotting, we previously reported that plasticity in the main olfactory bulb (MOB) underlies aversive olfactory learning. Since long-term potentiation (LTP) observed in the hippocampus is believed to be a cellular substrate for aspects of memory, we attempted to induce LTP in the MOB. Using brain slices containing the MOB, we found that five tetani of the lateral olfactory tract evoked LTP that was blocked by the N-methyl-d-aspartate (NMDA) receptor antagonist AP5. Although three tetani induced no significant changes in control slices, with noradrenaline (NA) application they produced clear LTP (NA-mediated LTP), which was not dependent on NMDA receptors. NA's facilitating effect on LTP induction was blocked by the β-adrenoceptor antagonist timolol but not by the α-adrenoceptor antagonist phentolamine, and was mimicked by the β-adrenoceptor agonist isoproterenol. The l-type calcium channel blocker nifedipine completely blocked LTP as well as NA-mediated LTP. In addition, we found that aversive olfactory learning was impaired by β-adrenoceptor antagonist, timolol but not by α-adrenoceptor antagonist, phentolamine, and only odor training established olfactory learning by isoproterenol infusion. Moreover, we found that nifedipine but not AP5 prevented olfactory learning formation. These common properties provided evidence for neural correlates between NA-mediated LTP aversive olfactory learning in young rats.     

The olfactory conditioning in the early postnatal period stimulated neural stem/progenitor cells in the subventricular zone and increased neurogenesis in the olfactory bulb of rats

The olfactory memory acquired during the early postnatal period is known to be maintained for a long period, however, its neural mechanism remains to be clarified. In the present study, we examined the effect of olfactory conditioning during the early postnatal period on neurogenesis in the olfactory bulb of rats. Using the bromodeoxyuridine-pulse chase method, we found that the olfactory conditioning, which was a paired presentation of citral odor (conditioned stimulus) and foot shock (unconditioned stimulus) in rat pups on postnatal day 11, stimulated the proliferation of neural stem/progenitor cells in the anterior subventricular zone (aSVZ), but not in the olfactory bulb, at 24 h after the conditioning. However, the number of newborn cells in the olfactory bulb was increased at 2 weeks, but not 8 weeks, after such conditioning. Neither the exposure of a citral odor alone nor foot shock alone affected the proliferation of neural stem/progenitor cells in the aSVZ at 24 h after and the number of newborn cells in the olfactory bulb at 2 weeks after. The majority of newborn cells in the olfactory bulb of either the conditioned rats or the unconditioned rats expressed the neural marker NeuN, thus indicating that the olfactory conditioning stimulated neurogenesis in the olfactory bulb. These results suggest that olfactory conditioning during the early postnatal period temporally stimulates neurogenesis in the olfactory bulb of rats.     

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.     

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.     

Olfactory modulation by dopamine in the context of aversive learning

The need to detect and process sensory cues varies in different behavioral contexts. Plasticity in sensory coding can be achieved by the context-specific release of neuromodulators in restricted brain areas. The context of aversion triggers the release of dopamine in the insect brain, yet the effects of dopamine on sensory coding are unknown. In this study, we characterize the morphology of dopaminergic neurons that innervate each of the antennal lobes (ALs; the first synaptic neuropils of the olfactory system) of the moth Manduca sexta and demonstrate with electrophysiology that dopamine enhances odor-evoked responses of the majority of AL neurons while reducing the responses of a small minority. Because dopamine release in higher brain areas mediates aversive learning we developed a naturalistic, ecologically inspired aversive learning paradigm in which an innately appetitive host plant floral odor is paired with a mimic of the aversive nectar of herbivorized host plants. This pairing resulted in a decrease in feeding behavior that was blocked when dopamine receptor antagonists were injected directly into the ALs. These results suggest that a transient dopaminergic enhancement of sensory output from the AL contributes to the formation of aversive memories. We propose a model of olfactory modulation in which specific contexts trigger the release of different neuromodulators in the AL to increase olfactory output to downstream areas of processing.     

Dopamine and serotonin receptors mediating contractions of the snail,Helix pomatia,salivary duct

The combination of high performance liquid chromatography, bioassay and immunocytochemistry was applied to study the regulation of the salivary duct muscle of the snail, Helix pomatia. The major function of the duct appears to be to propel the saliva toward the buccal cavity during feeding. It has been established that serotonin and dopamine applied exogenously mimic the effect on the duct exerted by the stimulation of the salivary nerve. Immunohistochemistry revealed the presence of serotonin, but not dopaminergic nerve elements in the nerve and along the duct surface. However, both serotonin (14.9-15.5 pmol/mg) and dopamine (0.38-0.58 pmol/mg), as well as the synthesizing enzymes (tyrozine hydroxylase 0.28 pmol/mg tissue/h and DOPA 0.32 nmol synthesized DA/mg tissue/h) could regularly be assayed in the salivary duct by high performance liquid chromatography. When released following the stimulation of the salivary nerve, both monoamines were shown to interact with distinct membrane receptors. Dopamine elicited a sustained increase of the muscle tone in concentration-dependent manner (K(d)=1.5 microM). Mammalian D(1) receptor antagonist flupenthixol and fluphenazine attenuated, whereas the D(1) receptor agonist SKF-38393 mimicked the effect elicited by exogenous dopamine. Serotonin had a double effect on the salivary duct: a relaxing and a contracting one with different K(d) values 76 nM and 2.4 microM, respectively. 5-HT(2) receptor antagonist ritanserin and ketanserin attenuated the serotonin-induced relaxation. In contrast 5-HT(3) antagonist metoclopramide and MDL2222 decreased and 5-HT(3) receptor agonist 1-(m-chlorophenyl)-biguanide mimicked the serotonin-induced contraction, suggesting that serotonin exerted its action on two different receptor subtypes. The release of radiolabeled serotonin and dopamine upon nerve stimulation was found to be Ca-dependent. Furthermore, the increase in serotonin concentration induced a decrease of the potency of dopamine to elicit sustained contraction.These results provide evidence for the transmitter role of serotonin and dopamine in salivary duct. It is concluded that receptors reveal a pharmacological profile related to vertebrate D(1), 5-HT(2) and 5-HT(3) receptor subtypes. Moreover, it was found that the process of conveying the saliva is modulated by an interaction of dopamine and serotonin.     

Increased dopamine after mating impairs olfaction and prevents odor interference with pregnancy

In rodents, social odor sensing influences female reproductive status by affecting neuroendocrine cascades. The odor of male mouse urine can induce ovulation or block pregnancy within 3 d post coitus. Females avoid the action of such olfactory stimuli after embryonic implantation. The mechanisms underlying these changes are unknown. Here we report that shortly after mating, a surge in dopamine in the mouse main olfactory bulb impairs the perception of social odors contained in male urine. Treatment of females at 6.5 d post coitus with a dopamine D2 receptor antagonist restores social odor sensing and favors disruption of pregnancy by inhibition of prolactin release, when administered in the presence of alien male urine odors. These results show that an active sensory barrier blocks social olfactory cues detrimental to pregnancy, consistent with the main olfactory bulb being a major relay through which social odor modulates reproductive status.     

Dopamine D(2) receptor activation modulates perceived odor intensity

Dopaminergic modulation affects odor detection thresholds and olfactory discrimination capabilities in rats. The authors show that dopamine D(2) receptor modulation affects odor discrimination capabilities in a manner similar to the modulation of stimulus intensity. Performance in a simultaneous odor discrimination task was systematically altered by manipulations of both odorant concentration and D(2) receptor activation (agonist quinpirole, 0.025-0.5 mg/kg; antagonist spiperone, 0.5 mg/kg). Rats' discrimination performance systematically improved at higher odor concentrations. Blockade of D(2) receptors improved performance equivalent to increasing odor concentration by 2 log units, whereas activation of D(2) receptors reduced odor discrimination performance in a dose-dependent manner. Bulbar dopamine release may serve a gain control function in the olfactory system, optimizing its sensitivity to changes in the chemosensory environment.     

Phencyclidine-induced head-twitch responses as 5-HT2 receptor-mediated behavior in rats

This study was designed to assess whether phencyclidine (PCP)-induced head-twitch was antagonized by ritanserin, a selective serotonin (5-HT2) receptor antagonist, in mice and rats to confirm the involvement of 5-hydroxytryptamine (5-HT) neurons in PCP actions in comparison with 5-methoxy-N,N-dimethyltryptamine (5-MeODMT)-induced behavior. PCP (7.5, 10 and 12.5 mg/kg, i.p.)-induced head-twitch was completely antagonized by ritanserin (1 mg/kg, s.c.) in mice and rats, and 5-MeODMT (2 and 4 mg/kg, i.p.)-induced head-twitch was also completely antagonized by ritanserin in mice. PCP and 5-MeODMT induced head-weaving in mice after ritanserin treatment, but this did not occur in rats. In rats, 5-MeODMT failed to induce head-twitch. These results suggest that PCP-induced head-twitch response in rats is developed via 5-HT2 receptors and it is a useful 5-HT2 receptor model, while 5-MeODMT-induced head-weaving in rats is developed via 5-HT1 receptors and is a useful 5-HT1 receptor model.     

Pharmacological characterization of an adenylyl cyclase-coupled 5-HT receptor in aplysia: comparison with mammalian 5-HT receptors

We attempted to identify compounds that are effective in blocking the serotonin (5-hydroxytryptamine, 5-HT) receptor(s) that activate adenylyl cyclase (AC) in Aplysia CNS. We call this class of receptor 5-HT(apAC). Eight of the 14 antagonists tested were effective against 5-HT(apAC) in CNS membranes with the following rank order of potency: methiothepin > metergoline approximately fluphenazine > clozapine > cyproheptadine approximately risperidone approximately ritanserin > NAN-190. GR-113808, olanzapine, Ro-04-6790, RS-102221, SB-204070, and spiperone were inactive. Methiothepin completely blocked 5-HT stimulation of AC with a K(b) of 18 nM. Comparison of the pharmacological profile of the 5-HT(apAC) receptor with those of mammalian 5-HT receptor subtypes suggested it most closely resembles the 5-HT(6) receptor. AC stimulation in Aplysia sensory neuron (SN) membranes was also blocked by methiothepin. Methiothepin substantially inhibited two effects of 5-HT on SN firing properties that are mediated by a cAMP-dependent reduction in S-K(+) current: spike broadening in tetraethylammonium/nifedipine and increased excitability. Consistent with cyproheptadine blocking 5-HT stimulation of AC, cyproheptadine also blocked the 5-HT-induced increase in SN excitability. Methiothepin was less effective in blocking AC-mediated modulatory effects of 5-HT in electrophysiological experiments on SNs than in blocking AC stimulation in CNS or SN membranes. This reduction in potency appears to be due to effects of the high ionic strength of physiological saline on the binding of this antagonist to the receptor. Methiothepin also antagonized AC-coupled dopamine receptors but not AC-coupled small cardioactive peptide receptors. In conjunction with other pharmacological probes, this antagonist should be useful in analyzing the role of 5-HT in various forms of neuromodulation in Aplysia.     

N-methyl-D-aspartate antagonist D-APV selectively disrupts taste-potentiated odor aversion learning.

Two experiments examined the effects of the competitive N-methyl-D-aspartate (NMDA) antagonist D-APV (D-2-amino-5-phosphonovalerate) on rats' ability to acquire potentiated aversions to the odor element of a taste-odor compound. In Experiment 1, pretreatment with D-APV (2.5 micrograms/side icv) caused stereospecific deficits in potentiated odor aversion learning but left simple taste and odor aversion learning intact. In Experiment 2, pretreatment with D-APV had no effect on rats' acquisition of an illness-based odor discrimination task. These results parallel those previously obtained using a noncompetitive NMDA antagonist (Robinson, Crooks, Shinkman, & Gallagher, 1989) and show that interference with NMDA receptors can selectively impair potentiated odor aversion learning. These results suggest that NMDA receptors play a critical role in some, but not all, forms of learning and memory.     

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.     

GABA(B)-mediated action in the frog olfactory bulb makes odor responses more salient

In the olfactory bulb, GABA(B) receptors are selectively located in the glomerular layer. A current hypothesis is that GABAergic inhibition mediated through these receptors would be, at least partly, presynaptic and would exerted by decreasing the release of the olfactory receptor neuron excitatory neurotransmitter. Here, we assessed, in the frog, the in vivo action of baclofen, a GABA(B) agonist, on single-unit mitral cell activity in response to odors. Local application of baclofen in the glomerular region of the olfactory bulb was shown to drastically affect mitral cell spontaneous activity, since they became totally silent. Moreover, under baclofen, mitral cells still responded to odors and still specified odor concentration increases through their temporal response patterns. The pharmacological specificity of the GABA(B) agonist action was confirmed by showing that saclofen, a GABA(B) antagonist, partly prevented the inhibitory action of baclofen and restored the initial rate of mitral cell spontaneous activity.The results show that GABA(B)-mimicked inhibition suppressed mitral cell spontaneous activity while odor responses were maintained. This suggests that olfactory receptor neurons partly drive spontaneous mitral cell activity. Moreover, the effect of GABA(B)-mediated inhibition was seen to be very close to that described previously for dopamine D(2) receptor-mediated inhibition. In conclusion, we propose that these two inhibitory mechanisms would offer the possibility to reduce or suppress mitral cell spontaneous activity so as to make their responses to odor especially salient.     

Dopamine reduces odor- and elevated-K(+)-induced calcium responses in mouse olfactory receptor neurons in situ

Although D2 dopamine receptors have been localized to olfactory receptor neurons (ORNs) and dopamine has been shown to modulate voltage-gated ion channels in ORNs, dopaminergic modulation of either odor responses or excitability in mammalian ORNs has not previously been demonstrated. We found that <50 microM dopamine reversibly suppresses odor-induced Ca2+ transients in ORNs. Confocal laser imaging of 300-microm-thick slices of neonatal mouse olfactory epithelium loaded with the Ca(2+)-indicator dye fluo-4 AM revealed that dopaminergic suppression of odor responses could be blocked by the D2 dopamine receptor antagonist sulpiride (<500 microM). The dopamine-induced suppression of odor responses was completely reversed by 100 microM nifedipine, suggesting that D2 receptor activation leads to an inhibition of L-type Ca2+ channels in ORNs. In addition, dopamine reversibly reduced ORN excitability as evidenced by reduced amplitude and frequency of Ca2+ transients in response to elevated K(+), which activates voltage-gated Ca2+ channels in ORNs. As with the suppression of odor responses, the effects of dopamine on ORN excitability were blocked by the D2 dopamine receptor antagonist sulpiride (<500 microM). The observation of dopaminergic modulation of odor-induced Ca2+ transients in ORNs adds to the growing body of work showing that olfactory receptor neurons can be modulated at the periphery. Dopamine concentrations in nasal mucus increase in response to noxious stimuli, and thus D2 receptor-mediated suppression of voltage-gated Ca2+ channels may be a novel neuroprotective mechanism for ORNs.     

Basolateral amygdala NMDA receptors are selectively involved in the acquisition of taste-potentiated odor aversion in the rat

In the taste-potentiated odor aversion (TPOA) paradigm, animals acquire a strong aversion to an odor that is followed by delayed intoxication only if a gustatory stimulus is presented with the odor during conditioning. Although previous work has shown that N-methyl-D-aspartate (NMDA) receptors in the basolateral nucleus of the amygdala (BLA) play a role in the acquisition of TPOA, the present study aimed at describing the process in which NMDA receptors in the BLA are involved during acquisition of TPOA. Male Long-Evans rats received intra-BLA infusions of the competitive NMDA receptor antagonist D,L-2-2-amino-5-phosphonovalerate (D-APV; 0.05 and 0.50 microg) immediately before or after the odor-taste conditioned stimulus (CS) presentation, or immediately before the test. Results showed that D-APV impaired acquisition of TPOA when infused before, but not after, the CS presentation, but did not affect retrieval. These results suggest that NMDA receptors of the BLA are involved in the formation of potentiation--by taste--of the olfactory memory trace, but not in the maintenance of this process.     

Changes in the categorization of appetitive and aversive events during postnatal development of the rat

Environmental stimuli are not clearly categorized into appetitive and aversive classes during the first postnatal week. Three- and 6-day-old rats are highly activated by nominally appetitive (milk infusions) and aversive (foot shock) stimuli. Both evoked the same generalized behavioral responses (rolling, curling). By 12 days of age, these stimuli were responded to differently; mouthing and probing occurred in response to milk while flinching and escape responses were observed to shock. The affective properties of mild shock were found to be hedonically opposite for 6- and 12-day-old pups. Six-day-old showed an acquired preference for an odor paired with shock which increased with intensity (.1–.5 mA) similar to that observed with milk infusions. An acquired odor aversion was displayed by 12 days old using these shock parameters. These results suggest that young rat pups may not differentiate many appetitive and aversive events behaviorally or affectively until the second postnatal week.