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Most mammals rely on chemosensory cues for individual recognition, which is essential to many aspects of social behavior, such as maternal bonding, mate recognition, and inbreeding avoidance. Both volatile molecules and nonvolatile peptides secreted by individual conspecifics are detected by olfactory sensory neurons in the olfactory epithelium and the vomeronasal organ. The pertinent cues used for individual recognition remain largely unidentified. Here we show that nonformylated, but not N-formylated, mitochondrially encoded peptides—that is, the nine N-terminal amino acids of NADH dehydrogenases 1 and 2—can be used to convey strain-specific information among individual mice. We demonstrate that these nonformylated peptides are sufficient to induce a strain-selective pregnancy block. We also observed that the pregnancy block by an unfamiliar peptide derived from a male of a different strain was prevented by a memory formed at the time of mating with that male. Our findings also demonstrate that pregnancy-blocking chemosignals in the urine are maternally inherited, as evidenced by the production of reciprocal sons from two inbred strains and our test of their urine’s ability to block pregnancy. We propose that this link between polymorphic mitochondrial peptides and individual recognition provides the molecular means to communicate an individual’s maternal lineage and strain.

Object recognition through tactile perception involves two elements: the shape of the object (macrogeometric properties) and the material of the object (microgeometric properties). Here we sought to determine the characteristics of microgeometric tactile representations regarding object recognition through tactile perception. Participants were directed to recognize objects with different surface materials using either tactile information or visual information. With a quantitative analysis of the cognitive process regarding object recognition, Experiment 1 confirmed the same eight concepts (composed of rules defining distinct cognitive processes) commonly generated in both tactile and visual perceptions to accomplish the task, although an additional concept was generated during the visual task. Experiment 2 focused only on tactile perception. Three tactile objects with different surface materials (plastic, cloth and sandpaper) were used for the object recognition task. The participants answered a questionnaire regarding the process leading to their answers (which was designed based on the results obtained in Experiment 1) and to provide ratings on the vividness, familiarity and affective valence. We used these experimental data to investigate whether changes in material attributes (tactile information) change the characteristics of tactile representation. The observation showed that differences in tactile information resulted in differences in cognitive processes, vividness, familiarity and emotionality. These two experiments collectively indicated that microgeometric tactile information contributes to object recognition by recruiting various cognitive processes including episodic memory and emotion, similar to the case of object recognition by visual information.

To explore the mechanism underlying the imitation of finger gestures, we devised a simple imitation task in which the patients were instructed to replicate finger configurations in two conditions: one in which they could see their hand (visual feedback: VF) and one in which they could not see their hand (non-visual feedback: NVF). Patients with left brain damage (LBD) or right brain damage (RBD), respectively, were categorized into two groups based on their scores on the imitation task in the NVF condition: the impaired imitation groups (I-LBD and I-RBD) who failed two or more of the five patterns and the control groups (C-LBD and C-RBD) who made one or no errors. We also measured the movement-production times for imitation. The I-RBD group performed significantly worse than the C-RBD group even in the VF condition. In contrast, the I-LBD group was selectively impaired in the NVF condition. The I-LBD group performed the imitations at a significantly slower rate than the C-LBD group in both the VF and NVF conditions. These results suggest that impaired imitation in patients with LBD is partly due to an abnormal integration of visual and somatosensory information based on the task specificity of the NVF condition.

Background. We have observed changes in body reactions during cooking, which is one of the treatment modalities used in occupational therapy. The perception of food-related odors during cooking may have behavioral effects on human activities through the activation of appetitive motivation. Objectives. We investigated whether odor components contained in seasonings could facilitate the human motor system and the specificity of this effect. Methods. The subjects were 72 healthy adults, randomly assigned to a water exposure group, a phenylethyl alcohol (PEA, pleasant rose-like odor) exposure group, and a Japanese soy sauce (Koikuchi Shoyu) exposure group (n=24 each). The subjects’ olfactory sense was stimulated by their sniffing of three different test tubes containing 5 ml of water, PEA, or Japanese soy sauce for 20 sec while they were seated. The modified Functional Reach Test (mFRT), which mimics a functional activity that is required in daily living and assesses a reliable measure of sitting balance, was performed prior to and immediately after the sniffing. Results. Sniffing the soy sauce increased the subjects’ mFRT scores. This facilitation effect was odorant-specific and was absent when the subjects were presented with water or PEA. Conclusions. Cooking interventions are aimed at improving tool-handling skills such as using knives and chopsticks. The results indicate that treatment interventions using odors of seasonings would be effective for improving subjects’ physical functions.

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.

Unilateral spatial neglect is a common neurological syndrome following predominantly right hemisphere damage, and is characterized by a failure to perceive and report stimuli in the contralesional side of space. To test the reference shift hypothesis that contralesional spatial neglect in right-brain-damaged patients is attributed to a rightward deviation of the egocentric reference frame, we measured the final angular position to which controls and left-side neglect patients actively turned their head toward the left in response to a verbal instruction given from each of three locations-right, left, and front-in two conditions, with and without visual feedback. When neglect patients were asked to "look straight ahead", they deviated about 30° toward the right in the eyes-open condition. However, the rightward deviation was markedly reduced in the eyes-closed condition. Regardless of visual feedback, there was no significant difference between controls and neglect patients in the final angular position of active head rotation when the verbal instruction came from the subject's left or front side; however, the final angular position was significantly smaller in the neglect patients than in the controls when the verbal instruction was given from the right. These results support the contention that cervico-vestibular stimulation during active head rotation restores spatial remapping and sensori-motor correlations and so improves neglect without affecting the position of the egocentric reference; however, once left-side neglect patients respond to verbal instruction from the right side, they are unable to disengage attention from the hemispace, and the performance of head rotation is disturbed.

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.

When female mice are mated, they form a memory to the pheromonal signal of their male partner. Several lines of evidence indicate that the neural changes underlying this memory occur in the accessory olfactory bulb (AOB) at the first stage of the vomeronasal system. The formation of this memory depends on the mating-induced release of noradrenaline in the AOB. In addition to noradrenaline, the neuropeptide oxytocin (OT) is also released within the central nervous system during mating. Because OT has been implicated in social memory and its receptors are expressed in the AOB, we hypothesized that OT might promote the strength of synaptic transmission from mitral to granule cells in the AOB. To test this hypothesis, we analyzed the lateral olfactory tract-evoked field potential that represents the granule cell response to mitral cell activation and its plasticity in parasagittal slices of the AOB. Of the 10-, 20-, 50-, and 100-Hz stimulations tested, the 100-Hz stimulation was optimal for inducing long-term potentiation (LTP). OT paired with 100-Hz stimulation that only produced short-term potentiation enhanced LTP induction in a dose-dependent manner. OT-paired LTP was blocked by both the selective OT antagonist desGly-NH 2 ,d(CH 2 ) 5 [Tyr(Me) 2 ,Thr 4 ]-ornithine vasotocin and the N -methyl- d -aspartate (NMDA) receptor antagonist dl -2-amino-5-phosphonovaleric acid. These results indicate that OT can function as a gate to modulate the establishment of NMDA receptor-dependent LTP at the mitral-to-granule cell synapse in the AOB.

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.

We investigated how simultaneous manipulation of suckling and i.c.v. injection of nitric oxide (NO)-related pharmacological agents affect Fos activation in the hippocampus and cortex of lactating rats. Dams received drugs or vehicle injection just prior to reunion with their pups following a 12 h separation and Fos protein activation was examined after 2 h of suckling. In conscious dams, sodium nitroprusside (SNP, an NO donor) significantly activated Fos expression in the hippocampus and cortex compared with vehicle or an NO synthase inhibitor. Urethane anaesthesia abolished Fos expression in the cortex, but not in the hippocampus following simultaneous SNP injection and resumption of suckling. Fos activation in the hippocampus and cortex was significantly attenuated when SNP was injected alone (without dam-pups reunion after separation). Coincident injection of SNP with a NO synthase inhibitor abolished all Fos activation in the hippocampus and cortex. In chronically lactating rats (without any interruption of suckling) SNP injection failed to activate any Fos expression. These observations underscore the importance of stimulus modality in revealing the dynamics of NO-mediated neuronal function within a discrete tissue entity such as the hippocampus or the cortex, which may be relevant to other regions of the brain.

Glial cells regulate some neural functions which depend on the homeostatic maintenance of extracellular calcium within narrow physiological ranges. In this study, the presence of microglia-specific ionized calcium binding adaptor molecule 1 (Iba1) was examined in the mouse olfactory bulb. A heterogenous pattern of Iba1-positive cells expression was observed between the main and accessory olfactory bulbs (MOB and AOB, respectively). While Iba1 was almost uniformly expressed among the laminae of the MOB, its expression showed spatial variations from the anterior to the posterior regions of the AOB. Double immunofluorescence was used to confirm that Iba1 is not expressed in astrocytes which stained for glial fibrillary acidic protein. Since Iba1 may mediate calcium signals in microglia, the observations suggest a potential involvement of Iba1 and hence microglia in olfactory bulb function and/or homeostasis. Together with our previous observation, this provides further support of a bulbar neuron–glia system of potential physiological significance.

An electrophysiological study was performed with mice lacking complexin II, a presynaptic protein. The long-term potentiation (LTP) by high-frequency stimulation, recorded in the hippocampal CA1 area, was decreased in complexin II-lacking mice (CPXII KO mice). The overall postsynaptic currents elicited by low frequency stimulation on the Schaffer collateral/commissural fibers in the hippocampal CA1 pyramidal cells were not different between wild-type and mutant mice. Excitatory postsynaptic currents (EPSCs) recorded in the presence of 50 microM bicuculline and inhibitory postsynaptic currents (IPSCs) recorded in the presence of 50 microM AP-5 (DL-2-amino-5-phosphonopentanoic acid) + 30 microM CNQX (6-cyano-7-nitroquinoxaline-2,3-dione) were also identical between wild-types and mutants. Furthermore, the EPSCs following repetitive stimulation (10 Hz) in CPXII KO mice did not show any difference with wild-types. These findings suggest that complexin II does not play a crucial role in ordinary neural transmission, short-term synaptic plasticity or synaptic transmission during high-frequency repetitive stimulation. Therefore, the protein is thought to be involved in the LTP process following tetanic stimulation, including the induction and/or maintenance of the LTP.

The exact nature of how nitric oxide (NO) acts in the regulation of milk ejection during lactation is not clearly understood at the moment. In this study, we have examined the effect of drugs which spontaneously release NO (sodium nitroprusside, SNP) or inhibit the NO synthase (NOS) enzyme (Nω-nitro- l -arginine, l -NA) on the activity of some hypothalamic and functionally associated nuclei using Fos expression as an index of neuronal activation. Lactating rats received intracerebroventricular injection of SNP, l -NA or vehicle (saline) just before they were reunited with their pups after a 12-h period of separation and allowed to suckle for 2 h. The difference in the total pup body weight before and after the period of suckling was used as a functional end-point of milk transfer. Central injection of SNP in conscious rats significantly inhibited Fos expression in the paraventricular nucleus (PVN), supraoptic nucleus (SON), periventricular and preoptic nuclei and also decreased pup body weight compared with saline- or l -NA-injected rats. Urethane-anesthetized animals, compared with their conscious counterparts, showed increased Fos expression in the PVN and SON. However, Fos expression in the PVN of the anesthetized animals was attenuated by l -NA injection compared with SNP and saline injection. Taken together with an earlier finding that SNP disrupts the milk ejection burst of oxytocinergic neurons, these observations suggest that NO may act within the neuron(s) possibly to alter the mechanism(s) regulating the periodic neuronal burst activity during lactation.

Female mice develop a long-lasting olfactory recognition memory of a partner male at the first relay in the vomeronasal system. In this study the synaptic plasticity relevant to this phenomenon was examined at reciprocal dendrodendritic synapses in the accessory olfactory bulb of female mice by electron microscopy. The size of asymmetrical excitatory synapses (mitral/tufted to granule cells) of the reciprocal synapses was significantly larger in the group of female mice which were subjected to a treatment intended to induce olfactory memory formation than in the control group. It is suggested that olfactory memory formation is associated with a conformational change at the level of synaptic structure of the 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.

The gaseous neurotransmitter molecule nitric oxide (NO) has recently generated a lot of interest on account of its possible physiological role in several models of learning and memory, both in vitro and in vivo. The presence of its synthesizing enzyme has been reported in the granule cell and external plexiform layers of the accessory olfactory bulb (AOB) in mice and rats. We have tested the effect of different doses of the nitric oxide synthase inhibitor, N ω -nitro- l -arginine applied locally and peripherally, on the formation of olfactory recognition memory in the context of pregnancy block in mice. Local infusions of 5, 10, and 40 nmol of the NOS inhibitor into the AOB failed to prevent memory formation of the stud male without affecting the effectiveness of the strange male to induce pregnancy block. Peripheral administration of the NOS inhibitor produced a pregnancy block rate that was linearly related to the dose regardless of whether or not exposure to the familiar or no male subsequently followed. This suggests that the effect of peripheral administration of the NOS inhibitor on memory formation could not be assessed using this experimental paradigm. The observations made in this study do not enable us to envisage any critical or primary physiological role for NO in this memory model. Its role, at best, may be modulatory and not obligatory.

Objective In the United States and European countries, the University of Pennsylvania Smell Identification Test (UPSIT), the Quick Smell Identification Test (Q-SIT), a shorter version of the UPSIT, and “Sniffin’ Sticks” are widely used for testing olfaction. The odor stick identification test for Japanese (OSIT-J) has been manufactured in Japan. This was followed by the development of “Open Essence,” consisting of cards that make use of sealed odorants. This study examined the usefulness of Open Essence in young volunteers with normal olfaction. Method The participants were 176 medical students in their clinical clerkships at the Department of Otolaryngology, Kochi Medical School Hospital. There were 111 males and 65 females, with a median age of 24 years (range 22–42 years). The olfactory function of all participants was confirmed as normal by screening with T&T olfactometry. Before beginning Open Essence, the subjects were asked to answer a questionnaire on their olfaction and habits. Results Females had significantly better smell identification than males. Of the 12 odorants, “perfume” and “cooking gas” were difficult to identify, because perfume involves different imagery and the compounds that give the smell of gas are readily volatilized. In contrast, all participants recognized “curry” and “menthol,” probably because they are received as both somatosensory stimuli and smell. Conclusions These results suggest that, with improvement, Open Essence might be used for testing olfaction in Japanese subjects.

When female mice are mated, they form a memory to the pheromonal signal of their male partner. The neural mechanisms underlying this memory involve changes at the reciprocal dendrodendritic synapses between glutamatergic mitral cells and gamma-aminobutyric acid (GABA)-ergic granule cells in the accessory olfactory bulb (AOB). Blockade of GABA(A) receptors in the AOB leads to the formation of an olfactory memory. In an attempt to disrupt memory formation at mating, we used local infusions of the GABA(A) receptor agonist muscimol into the AOB during the critical period for memory formation. Muscimol across a wide range of doses (1-1000 pmol) did not prevent memory formation. The resistance of this memory to GABA(A) receptor activation may reflect the complexity of synaptic microcircuits in the AOB.

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.

Mice have an olfactory (pheromone) recognition memory located at the first relay in the sensory system. It is acquired with one-trial learning, contingent upon norepinephrine activation at mating, and lasts for several weeks. The mechanism involves Hebbian (association-dependent) changes in synaptic efficacy at dendrodendritic synapses in the accessory olfactory bulb. As a result of this memory, males made familiar by mating are recognized by the females, thereby mitigating pregnancy block. Such a memory function is biologically important to the female, as it is required to sustain pregnancy in the presence of her stud male's odors.

Objective To investigate the differences in regions of brain activation in response to olfactory stimulation by functional magnetic resonance imaging in conditions of prior warning of an odor and without. Methods Participants were 17 normal right-handed volunteers; 8 participants received prior warning of the odor (informed condition) and 9 participants were not pre-warned (naive condition). The odorant used was isovaleric acid. Results In the informed condition with prior warning, activation was observed in the putamen extending to the insula, amygdala, and inferior frontal gyrus, and there was instant reification of the odor, while in the naive condition without prior warning, activation was observed in the anterior cingulate cortex, entorhinal cortex, putamen and inferior frontal gyrus, and recognition of the odor was difficult. Conclusions These results suggest that the condition prior to olfactory stimulation, i.e., with or without prior warning, can affect recognition and regions of brain activation in response to olfactory stimulation using isovaleric acid. Differences in recognition and regions of brain activation between both conditions could be associated with response latencies, or degree of attention, expectation and/or concentration.

One context of olfactory learning that has been investigated in some detail concerns the memory, established at mating, and formed by the female mouse to the odours (pheromones) of the mating male. This olfactory memory is vital for mitigating pregnancy block that might otherwise be induced by his pheromones (Keverne and Rosser, 1986). Pheromones from an unfamiliar male, for which no memory has been formed, activate the vomeronasal system with their receptors in the vomeronasal organ, thereby initiating a neuroendocrine reflex that suppresses prolactin secretion from the pituitary (Keverne, 1983). The removal of luteotrophic support results in a fall in progesterone levels and a return to oestrus. Therefore, it has been hypothesized that the pheromonal memory functions as a gate to suppress or modulate the specific pheromonal signal (Brennan et al., 1990; Kaba and Nakanishi, 1995; Brennan and Keverne, 1997; Brennan, 2001). The pheromonal memory is acquired with one trial learning, depends upon mating and lasts for several weeks (Keverne and de la Riva, 1982; Kaba et al., 1988). The neural changes underlying memory formation occur in the accessory olfactory bulb (AOB), the first relay in the vomeronasal system, independently of the main olfactory system and the hippocampus (Brennan et al., 1990; Kaba and Nakanishi, 1995; Brennan and Keverne, 1997). Microcircuits in the AOB include the prominent reciprocal dendrodendritic synapse between mitral cells, a single class of projection neurons and granule cell interneurons. Glutamate released from mitral cell dendrites activates the dendrites of granule cells, which in turn mediate GABAergic dendrodendritic inhibition back onto mitral cell dendrites (Jia et al., 1999; Taniguchi and Kaba, 2001). This feedback inhibition at the reciprocal synapses regulates mitral cell activity (Jia et al., 1999; Taniguchi and Kaba, 2001). The formation of the pheromonal memory requires the association of the pheromonal and the mating signals in the AOB. The mating signal is conveyed by noradrenergic projections from the locus coeruleus. Artificial vaginocervical stimulation (Rosser and Keverne, 1985) or mating (Brennan et al., 1995) promotes the release of noradrenaline (NA) in the AOB. Blockade of α-adrenergic receptors in the AOB immediately after mating prevents the formation of the pheromonal memory (Kaba and Keverne, 1988), as does removal of noradrenergic innervation of the AOB prior to mating (Rosser and Keverne, 1985). Furthermore, memory formation is associated with neurochemical and morphological changes at the mitral–granule cell reciprocal synapses (Brennan et al., 1995; Matsuoka et al., 1997, 2004). Despite advances such as these, however, an important void in our knowledge of electrophysiological aspects has remained: a longlasting increase in synaptic strength, known as long-term potentiation (LTP), has been little investigated. Moreover, the cellular and synaptic mechanisms underlying noradrenergic modulation of pheromonal learning are also unknown. To address these questions, we have carried out a series of experiments in AOB slices. Induction of LTP at the mitral-to-granule cell synapse in the AOB

We investigated a model of long-term memory in which the female mouse establishes pheromonal memory of its partner at mating. We examined the reciprocal synapses of the accessory olfactory bulb and found that pheromonal memory was associated with morphological changes in excitatory synapses in the early phase of memory acquisition and by changes in inhibitory synapses in the late phases of memory persistence. After extinction of pheromonal memory, these morphological changes were no longer present. These findings suggest that the persistence of pheromonal memory is associated with continuous and dynamic changes in the morphological plasticity of reciprocal synapses in the accessory olfactory bulb.

The exact role of leptin in fasting has not been completely elucidated. To determine whether leptin can act in fasting to influence plasma estrogen levels and nitric oxide synthase reactivity in food regulating centers of the brain, we fasted female rats for 4 days and treated them i.p. with vehicle or 100 microg of recombinant mouse leptin as 1 ml on the 3rd and 4th day twice daily (10.00 and 17.00 h). Proestrus blood was collected at 10.00, 14.00, 18.00 and at 22.00 h, plasma obtained and assayed for estrogen (E2) and leptin levels. Verification of ovulation occurrence was by examining the oviduct for extruded ovum. The rat brains were removed and processed for nitric oxide synthase reactivity in the ventromedial hypothalamus (VMH) and arcuate nucleus (ARC) using NADPH-diaphorase histochemistry, a marker for neurons expressing NOS enzyme. Leptin effect on dependable variables such as food intake, water intake and body weight gain was also investigated. Four days fasting significantly decreased body weight, estrogen and postfast leptin levels, nitric oxide reactivity in the VMH and ARC nucleus and stopped ovulation in many (4 out of 5) rats fasted and given vehicle. Leptin treatment significantly increased plasma estrogen and postfast leptin levels, restored ovulation in many (4 out of 5) rats and increased nitric oxide reactivity in the VMH and ARC. Leptin significantly inhibited food intake, water intake and gain in body weight during recommenced feeding. These observations suggest that leptin could act in the pituitary-ovarian axis during fasting to improve reproductive function by partly stimulating estrogen secretion.

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.

Oxytocin (OT) modulation of synaptic transmission between olfactory bulb neurones has been implicated in the induction of maternal behaviour, but the mechanism of action is unknown. We examined the action of OT on gamma-aminobutyric acid(A) (GABA(A)) receptor-mediated spontaneous inhibitory postsynaptic currents (sIPSCs) in cultured mitral/tufted (M/T) cells with the use of whole-cell patch-clamp recordings. OT reversibly reduced the frequency of sIPSCs without affecting the amplitudes. The effect of OT on sIPSCs was mimicked by the OT receptor agonist [Thr(4), Gly(7)]-OT in a reversible manner and blocked by the OT receptor antagonist desGly-NH(2)(9), d(CH(2))(5)-[Tyr(Me)(2), Thr(4)]-ornithine-vasotocin. OT has no effect, however, on the membrane currents evoked by exogenous application of GABA. These results demonstrate that OT depresses GABA(A) receptor-mediated sIPSCs in M/T cells by a presynaptic mechanism.

Glutamine synthetase (GS) critically regulates the metabolism of glutamate and γ-amino butyric acid (GABA), which mediate synaptic plasticity in the olfactory bulb. In this study, GS immunolocalization in the mouse olfactory bulb was examined. The main and accessory subdivisions of the olfactory bulb possess GS-positive cells and processes in the plexiform-, the mitral- and the granule cell layers. GS has been demonstrated to show a predominantly astrocytic localization; its presence in the cell layers implicated in glutamatergic and GABAergic function therefore suggests that bulbar synaptic plasticity in mice may be regulated by astroglia and, together with other lines of evidence, point to the possibility of a functional astroglia–neuron system in the mouse olfactory bulb.

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.

Some forms of olfactory learning require intact noradrenergic terminals in the olfactory bulb that originate from the locus coeruleus. To clarify the action of noradrenergic inputs on the dendrodendritic interaction between mitral and granule cells in the rat olfactory bulb, we analyzed field potentials in the granule cell layer of the olfactory bulb evoked by paired-pulse stimulation of the lateral olfactory tract before and after the activation of the locus coeruleus. Locus coeruleus activation by glutamate injection in the vicinity of the nucleus changed only the test response without any effect on conditioning response. Paired-pulse inhibition measured from the ratio of test response amplitude to conditioning response amplitude was significantly depressed immediately after locus coeruleus activation. Conversely, 2 min later, paired-pulse inhibition was significantly potentiated. The significant potentiation of inhibition lasted for several minutes. The depression-potentiation sequence of paired-pulse inhibition was blocked by infusion of timolol, a beta-antagonist, into the olfactory bulb, in a dose-dependent manner, but not by infusion of phentolamine, an alpha-antagonist. Infusion of isoproterenol, a beta-agonist, into the bulb mimicked the depression of paired-pulse inhibition by locus coeruleus activation. These results suggest that glutamate activation of the locus coeruleus produces a depression-potentiation sequence in granule cell-mediated feedback inhibition onto mitral cells in the olfactory bulb through beta-adrenergic receptors.

Infusions of a nitrosothiol, (±) S -nitroso- N -acetylpenicillamine or l -arginine into the accessory olfactory bulb (AOB) are capable of causing memory or learning-related synaptic plasticity in female mice. This is consistent with the ability of nitric oxide (NO), released spontaneously or by activation of the endogenous synthesizing system, to enhance a long-lasting increase in gain of AOB synapses; resulting in memory formation for male pheromones without mating. Hemoglobin, a NO scavenger, prevented this memory suggesting, in part, an extracellular action of NO in this functional plasticity.

We examined the effect of nitric oxide (NO) on the process of parturition in rats. Subcutaneous injection of the NO donor sodium nitroprusside (SNP) in late pregnancy prolonged the total parturition time. The effect of N omega-nitro-L-arginine was not significantly different from that of saline. Intracerebroventricular injection of SNP in parturient rats delayed the progress of parturition. Both modes of SNP treatment also inhibited expression of maternal behavior. Central injection of oxytocin (OXT) with SNP failed to reduce parturition time significantly, but intrapartum, not postpartum, maternal behaviour was restored. These observations suggest that NO interferes with the release of OXT within the brain, hence affecting the initiation of maternal behaviour, and may also impair oxytocin secretion from the neurohypophysis.

Field potentials evoked in the accessory olfactory bulb (AOB) of the mouse by a vomeronasal organ (VNO) volley were analyzed with current source-density (CSD) techniques. The potentials were recorded with an electrode oriented in the direction perpendicular to the laminae of the centre of the AOB in five tracks of equal spatial lattice for identical coordinates. There were little or no differences in the amplitude or time course of the potentials at equal depth among the five tracks. The CSD profile from a one-dimensional CSD analysis corresponded closely with that of a three-dimensional CSD analysis of a central track in the AOB. Furthermore, VNO-evoked field potentials were recorded in the AOB external plexiform layer while an electrode was being moved in the direction parallel to this layer. The potentials recorded at adjacent points were nearly identical and hence the related one-dimensional CSD calculations revealed no net currents along this layer. These results indicate that a one-dimensional CSD analysis is applicable to the mouse AOB.

The complexity and inaccessibility of the mammalian brain prevent the localization and description of memory traces and the definition of the processes that produce memories. The model reviewed here is the olfactory recognition memory formed to male pheromones by a female mouse at mating. The memory trace has been localized to the reciprocal dendrodendritic synapse between mitral cells and granule cells in the accessory olfactory bulb. An increase in noradrenaline after mating reduces inhibitory transmission of gamma-aminobutyric acid (GABA) from the granule cells to mitral cells and induces an olfactory memory of pheromones present at mating. Recent work has shown that the activation of mGluR2, a metabotropic glutamate receptor, localized at granule cell dendrites suppresses the GABA inhibition of the mitral cells and permits the formation of a specific olfactory memory that faithfully reflects the memory formed at mating. This simple olfactory memory may provide an excellent model system with which to investigate the molecular mechanisms of the synaptic plasticity involved in learning and memory.

Female mice form an olfactory memory of male pheromones at mating; exposure to the pheromones of a strange male after that mating will block pregnancy. The formation of this memory is mediated by the accessory olfactory system, in which an increase in norepinephrine after mating reduces inhibitory transmission of gamma-aminobutyric acid from the granule cells to the mitral cells. This study shows that the activation of mGluR2, a metabotropic glutamate receptor that suppresses the gamma-aminobutyric acid inhibition of the mitral cells, permits the formation of a specific olfactory memory without the occurrence of mating by infusion of mGluR2 agonists into the female's accessory olfactory bulb. This memory faithfully reflects the memory formed at mating.

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.

Electrical stimulation of the rat A1 noradrenergic region produced orthodromic inhibition (34%), excitation (23%) or excitation-inhibition sequence (25%) of the spontaneous activity of phasically firing units in the paraventricular nucleus of the hypothalamus (PVH). Excitatory responses could be distinguised into two types on the basis of their latency and duration. Both the primary and post-excitatory inhibitory responses were significantly attenuated by intravenous injection of propranolol, a β-adrenoceptor antagonist, whereas neither type of excitatory response was affected.

Seventy neurons in the ventrolateral medulla oblongata were antidromically activated by electrical stimulation of the preoptic and anterior hypothalamic area (POA/AHA) in female rats under urethane anesthesia. These identified cells were located within and adjacent to the nucleus reticularis lateralis and could be readily distinguished into at least two types of neurons, designated as ‘fast’ and ‘slow’ cells, on the basis of their waveform and conduction velocity.

Estradiol benzoate administered to ovariectomized female rats significantly elevated the mean spontaneous firing rate and frequency of successful antidromic propagation into the somatodendritic spike and significantly reduced the strength of post-stimulus inhibition in presumed A1 noradrenergic neurons projecting directly to the preoptic and anterior hypothalamic area. The occurrence of both antidromic spikes and post-stimulus inhibition of the majority of these neurons was completely abolished by the injection of 6-hydroxydopamine but not by 5, 7-dihydroxytryptamine directly into the medial forebrain bundle.

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.

Noradrenaline (NA), but not clonidine applied microiontophoretically, inhibited the firing of neurons in the rat A1 noradrenergic region, whereas intravenous clonidine dose-dependently inhibited their firing (ED50 = 18.3 micrograms/kg). The NA-induced inhibition was blocked by the beta-antagonist timolol, but not by the alpha-antagonists phentolamine or piperoxane. These results suggest that neurons in the A1 region possess beta-adrenoceptors rather than alpha-adrenoceptors near their cell bodies, and that systemically administered clonidine acts indirectly on these cells.