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Epigenetic mechanisms play an important role in memory formation and synaptic plasticity. Specifically, histone-associated heterochromatin undergoes changes in structure during the early stages of long-term memory formation. In keeping with the classical conditioning paradigm, young rats have been shown to exhibit aversion to an odor stimulus initially presented during foot shock. We previously showed that synaptic plasticity at the dendrodendritic synapses between mitral and granule cells in the olfactory bulb (OB) underlies this aversive olfactory learning. However, the epigenetic mechanisms involved are not well characterized. Therefore, we examined whether intrabulbar infusion of trichostatin A (TSA), a histone deacetylase inhibitor, facilitates olfactory learning in young rats. TSA infusion during odor-shock training enhanced a conditioned odor aversion in a dose-dependent manner and prolonged the learned aversion. Western blot and immunohistochemical analyses showed that the level of histone H4 acetylation significantly increased until 4 h after odor-shock training in both mitral and granule cells in the OB, whereas histone H3 acetylation returned to the control level at 2 h after the training. We also obtained evidence that TSA infusion elevated acetylation of histone H4 or H3. Furthermore, in vitro electrophysiological analysis using slices of the OB revealed that application of TSA significantly enhanced the long-term potentiation induced in synaptic transmission from mitral to granule cells at dendrodendritic synapses. Taken together, these results provide evidence that histone H4 and H3 acetylation in the OB is an epigenetic mechanism associated with aversive olfactory learning in young rats.

Tunicamycin (TM) induces endoplasmic reticulum (ER) stress and inhibits N-glycosylation in cells. ER stress is associated with neuronal death in neurodegenerative disorders, such as Parkinson's disease and Alzheimer's disease, and most patients complain of the impairment of olfactory recognition. Here we examined the effects of TM on aversive olfactory learning and the underlying synaptic plasticity in the main olfactory bulb (MOB). Behavioral experiments demonstrated that the intrabulbar infusion of TM disabled aversive olfactory learning without affecting short-term memory. Histological analyses revealed that TM infusion upregulated C/EBP homologous protein (CHOP), a marker of ER stress, in the mitral and granule cell layers of MOB. Electrophysiological data indicated that TM inhibited tetanus-induced long-term potentiation (LTP) at the dendrodendritic excitatory synapse from mitral to granule cells. A low dose of TM (250nM) abolished the late phase of LTP, and a high dose (1μM) inhibited the early and late phases of LTP. Further, high-dose, but not low-dose, TM reduced the paired-pulse facilitation ratio, suggesting that the inhibitory effects of TM on LTP are partially mediated through the presynaptic machinery. Thus, our results support the hypothesis that TM-induced ER stress impairs olfactory learning by inhibiting synaptic plasticity via presynaptic and postsynaptic mechanisms in MOB.

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.

Reciprocal dendrodendritic synapses between mitral and granule cells in the accessory olfactory bulb have been implicated in a specialized form of olfactory learning in mice, in which a female forms a memory to the pheromonal signal of the male that mates with her. Relatively little is known, however, about the mechanism of synaptic transmission at the reciprocal synapses. We analyzed synaptic currents generated in accessory olfactory bulb mitral cells in slice preparations with the patch-clamp technique in nystatin-perforated whole-cell configuration. A brief (5–20-ms) depolarizing voltage step from −70 to 0 mV applied to a single mitral cell evoked GABA A receptor-mediated inhibitory postsynaptic currents. The inhibitory postsynaptic currents persisted in the presence of tetrodotoxin, indicating that the inhibitory postsynaptic current in mitral cells can be elicited through purely dendritic interactions. The inhibitory postsynaptic currents were greatly enhanced by washout of extracellular Mg 2+ . In Mg 2+ -free solution, the N -methyl- D -aspartate (NMDA) receptor antagonist D , L -2-amino-5-phosphonovaleric acid greatly reduced the inhibitory postsynaptic currents, whereas the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-(1 H ,4 H )-dione (CNQX) slightly reduced them. These data demonstrate that NMDA receptors play an important role in the generation of dendrodendritic inhibition in mitral cells of the mouse accessory olfactory bulb.

The effects of electrical stimulation of the hypothalamus paraventricular nucleus on the spontaneous firing of mitral and granule cells in the main olfactory bulb were examined in ovariectomized female rats under urethane anaesthesia. High-frequency stimulation (0.5-1.0 mA, 10-20 pulses at 100 Hz) of the paraventricular nucleus produced inhibitory responses in 80% of mitral cells tested and excitatory responses in 74% of granule cells tested, with latencies ranging from 2 to 150 s. Both responses were blocked by infusions into the olfactory bulb of [d(CH2)5, Tyr(Me)2]ornithine-vasotocin (10 pmol), an oxytocin antagonist, and mimicked by intracerebroventricular infusions (0.2 or 0.4 nmol) or microiontophoretic applications of oxytocin but not by intracerebroventricular infusions of vasopressin (1 or 2 nmol). Infusions of 0.5% lignocaine, a local anaesthetic, into either the medial olfactory tract or the medial forebrain bundle failed to block the responses of mitral and granule cells to the stimulation. Unilateral transections at various levels between the bulb and the paraventricular nucleus also failed to block the responses. There were cases in which significant responses of mitral and granule cells to the stimulation required 60 or more pulses after the lignocaine infusions or transections, however. These results suggest that oxytocin originating in the hypothalamic paraventricular nucleus reaches the olfactory bulb following its release partly into the cerebrospinal fluid and acts to decrease olfactory processing.

The plexiform and granule cell layers of the female mouse accessory olfactory bulb, whose synaptic activities are modified by pheromonal inputs after mating, contain one of the highest densities of nitric oxide synthase in the brain. We tested the hypothesis that exogenous nitric oxide administration can, in principle, permit the formation of a specific pheromonal memory without mating by acting in synergy with bulbar neurotransmitter(s) to enhance long-lasting increase in gain of the mitral-granule cell dendrodendritic synapse. Two infusions of sodium nitroprusside (5 nmol; 0.5 microliters) into the accessory olfactory bulb activated recognition without mating. A single infusion produced no recognition. This memory is specific to the pheromones to which the females were exposed during sodium nitroprusside infusions because strange male pheromones evoked a significant pregnancy failure rate. Furthermore, the memory formation is dependent on coincident activation by pheromonal inputs and sodium nitroprusside infusions, since drug infusions in the absence of male pheromones permitted a significant pregnancy block on test exposure. The alpha-adrenergic antagonist phentolamine prevented a sodium nitroprusside-mediated memory formation. In females with depleted bulbar noradrenergic innervation by specific neurotoxin (6-hydroxydopamine) injection into the medial olfactory striae or the accessory olfactory bulb, sodium nitroprusside infusions failed to induce memory formation. The procedure itself apparently did not interfere with the occurrence of pregnancy. These results demonstrate that exogenous administration of nitric oxide can induce a pheromone-specific olfactory memory without mating, and that this memory is mediated, at least in part, by noradrenaline.

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 beta-adrenoceptor antagonist timolol but not by the alpha-adrenoceptor antagonist phentolamine, and was mimicked by the beta-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 beta-adrenoceptor antagonist, timolol but not by alpha-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.

To investigate the interaction between vomeronasal receptor neurons and accessory olfactory bulb neurons during pheromonal signal processing and specific synapse formation, partially dissociated rat vomeronasal receptor neurons were co-cultured with accessory olfactory bulb neurons. Between 7 and 14 days in co-culture, a few bundles of fibers from a spherical structure, termed the vomeronasal pocket, of cultured vomeronasal receptor neurons extended to the accessory olfactory bulb neurons. An optical recording of the intracellular Ca(2+) concentration was used to monitor the synaptic activation of cultured accessory olfactory bulb neurons. Electrical stimulation of the vomeronasal pocket between 7 and 14 days in co-culture had no effects on most of the cultured neurons tested, although it occasionally evoked weak responses in a small number of neurons. In contrast, vomeronasal pocket stimulation after 21 days in co-culture evoked clear calcium transients in a substantial number of cultured accessory olfactory bulb neurons. These responses of accessory olfactory bulb neurons were reversibly suppressed by the application of 6-cyano-7-nitroquinoxaline-2,3-dione; the calcium transients disappeared in most of the neurons and were diminished in the others. The application of d-2-amino-5-phosphonopentanoic acid partially affected the calcium transients, but blocked spontaneous calcium increases, which were observed repeatedly in accessory olfactory bulb-alone cultures. The application of both 6-cyano-7-nitroquinoxaline-2,3-dione and d-2-amino-5-phosphonopentanoic acid completely blocked the evoked calcium transients. These results suggest that functional glutamatergic synapses between vomeronasal receptor neurons and accessory olfactory bulb neurons were formed at around 21 days in co-culture.

Long-term memory formation requires both gene expression and protein synthesis. Phosphorylation of the transcription factor cyclic AMP response element-binding protein (CREB) is thought to be important in processes underlying long-term memory. To clarify the role of CREB in olfactory aversive learning in young rats, we carried out behavioral pharmacology and Western blot analyses. On postnatal day 11, oligodeoxynucleotides were infused directly into the bilateral olfactory bulbs through cannulae implanted prior to training in a classical conditioning paradigm with citral odor and foot shock. On the following day the odor preference test was performed. After training, saline-infused animals spent significantly shorter time over the citral odor zone. Infusion of CREB antisense oligodeoxynucleotides 6 h before or during training, however, prevented olfactory aversive learning without affecting memory retention 1 h after training. CREB scrambled oligodeoxynucleotides infusions had no effect on olfactory learning. When infused 6 h after training, none of oligodeoxynucleotides had an effect on time spent over the odor zone. Using Western blotting, we analyzed CREB in nuclear extracts obtained from the young rats after training. Marked increases in phosphorylated CREB were sustained from 10 to 360 min after the odor-shock pairing in animals which were subjected to both, in comparison with levels 30 min in animals which were subjected to odor only or no stimulation. Total CREB levels showed no differences among groups. Infusion of CREB antisense oligodeoxynucleotides significantly reduced the expression of phosphorylated and total CREBs in the olfactory bulb. These results show that the synthesis and phosphorylation of CREB are required for the acquisition of olfactory aversive learning in young rats, and that this requirement for the CREB signaling pathway has a critical time window.

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.

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.

On mating with a stud male, virgin female mice from an olfactory memory to this male which is essential to their reproductive success. Failure to form this memory results in the stud male being treated as strange, and hence, his pheromones block pregnancy. This study investigates the duration of the olfactory memory, and the factors which determine this. The results show that a single prolonged exposure to the male during mating forms a life-long olfactory memory trace unless pregnancy ensues. In the event of pregnancy the olfactory memory fades significantly faster, an effect which can be replicated by implants of oestradiol in non-pregnant females. Anatomical studies indicate that neurogenesis of the vomeronasal receptors is enhanced during pregnancy, an event which we interpret as being important for the duration of this olfactory memory.

Female mice which have mated and are subsequently exposed to the odour (pheromones) of a strange male undergo hormonal changes resulting in a block to their pregnancy. The fact that the stud male's odours can also block pregnancies, that is other than his own, implies the formation of a memory or some form of recognition process by the female for this male's pheromones at the time of mating. The purpose of this study was to evaluate the effect of microinfusions of drugs which interfere with neural transmission, into the accessory olfactory bulbs. This was carried out immediately after mating over a 4-h period during which the "memory" to the stud male's pheromones is formed. Infusions of the alpha-blocker, phentolamine, blocked the formation of the olfactory memory, while the GABA receptor blocker, bicuculline, itself blocked pregnancy, but was without effect on memory formation. Protein synthesis inhibition or calpain inactivation in the accessory bulb was without effect on memory formation at any of the doses used. These studies demonstrate that GABAergic transmitter blockade in the accessory olfactory bulb at the time of mating can prevent subsequent blastocyst implantation some 3 days later, while alpha-noradrenergic blockade can prevent the formation of an olfactory memory to the stud male.