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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.

The formation of mate recognition memory in mice is associated with neural changes at the reciprocal dendrodendritic synapses between glutamatergic mitral cell (MC) projection neurons and GABAergic granule cell (GC) interneurons in the accessory olfactory bulb (AOB). Although noradrenaline (NA) plays a critical role in the formation of the memory, the mechanism by which it exerts this effect remains unclear. Here we used extracellular field potential and whole-cell patch-clamp recordings to assess the actions of bath-applied NA (10 µM) on the glutamatergic transmission and its plasticity at the MC-to-GC synapse in the AOB. Stimulation (400 stimuli) of MC axons at 10 Hz but not at 100 Hz effectively induced N-methyl-d-aspartate (NMDA) receptor-dependent long-term potentiation (LTP), which exhibited reversibility. NA paired with subthreshold 10-Hz stimulation (200 stimuli) facilitated the induction of NMDA receptor-dependent LTP via the activation of α2-adrenergic receptors (ARs). We next examined how NA, acting at α2-ARs, facilitates LTP induction. In terms of acute actions, NA suppressed GC excitatory postsynaptic current (EPSC) responses to single pulse stimulation of MC axons by reducing glutamate release from MCs via G-protein coupled inhibition of calcium channels. Consequently, NA reduced recurrent inhibition of MCs, resulting in the enhancement of evoked EPSCs and spike fidelity in GCs during the 10-Hz stimulation used to induce LTP. These results suggest that NA, acting at α2-ARs, facilitates the induction of NMDA receptor-dependent LTP at the MC-to-GC synapse by shifting its threshold through disinhibition of MCs.

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.

Olfaction plays an important role in social recognition in most mammals. Central arginine vasopressin (AVP) plays a role in this olfaction-based recognition. The high level of expression of AVP receptors in the accessory olfactory bulb (AOB) at the first relay of the vomeronasal system highlights the importance of AVP signaling at this stage. We therefore analyzed the effects of AVP on the synaptic plasticity of glutamatergic transmission from mitral cells to granule cells in AOB slices from male mice. To monitor the strength of the glutamatergic transmission, we measured the maximal initial slope of the lateral olfactory tract-evoked field potential, which represents the granule cell response to mitral cell activation. AVP paired with 100-Hz stimulation that only produced short-term potentiation enhanced the induction of long-term potentiation (LTP) in a dose-dependent manner. AVP-paired LTP was blocked by the selective AVP receptor 1a (AVPR1a) antagonist, d(CH2)5[Tyr(Me)2]AVP (Manning compound), but not by the AVPR1b antagonist SSR149415, and it was mimicked by the selective AVPR1a agonist [Phe2, Ile3, Orn8]-vasopressin. We further examined the effect of AVP on the reciprocal transmission between mitral and granule cells by stimulating a mitral cell and recording the evoked inhibitory postsynaptic currents (IPSCs) from the same cell using conventional whole-cell patch-clamp techniques. AVP reduced the reciprocal IPSCs triggered by endogenous glutamate release from the excited mitral cell. These results suggest that AVP promotes the induction of LTP at the mitral-to-granule cell synapse via the activation of AVPR1a through an as-yet-to-be-determined mechanism in the AOB of male mice.

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.

[Purpose] To examine the ability of young and elderly individuals to control the timing and force of periodic sequential foot tapping. [Subjects and Methods] Participants were 10 young (age, 22.1 ± 4.3 years) and 10 elderly individuals (74.8 ± 6.7 years) who were healthy and active. The foot tapping task consisted of practice (stimulus-synchronized tapping with visual feedback) and recall trials (self-paced tapping without visual feedback), periodically performed in this order, at 500-, 1,000-, and 2,000-ms target interstimulus-onset intervals, with a target force of 20% maximum voluntary contraction of the ankle plantar-flexor muscle. [Results] The coefficients of variation of force and intertap interval, used for quantifying the steadiness of the trials, were significantly greater in the elderly than in the young individuals. At the 500-ms interstimulus-onset interval, age-related effects were observed on the normalized mean absolute error of force, which was used to quantify the accuracy of the trials. The coefficients of variation of intertap interval for elderly individuals were significantly greater in the practice than in the recall trials at the 500- and 1,000-ms interstimulus-onset intervals. [Conclusion] The elderly individuals exhibited greater force and timing variability than the young individuals and showed impaired visuomotor processing during foot tapping sequences.

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.

The throughput of information from the accessory olfactory bulb (AOB) to downstream structures is controlled by reciprocal dendrodendritic inhibition of mitral cells by granule cells. Given the high expression levels of mGluR2, a metabotropic glutamate receptor, in the AOB and the fact that the activation of mGluR2 permits the formation of a specific olfactory memory, we reasoned that mGluR2 might play an important role in regulating dendrodendritic inhibition. To test this hypothesis, we examined the effects of pharmacological and genetic manipulations of mGluR2 on synaptic responses measured from mitral or granule cells in slice preparations from 23- to 36-day-old Balb/c mice. To evoke dendrodendritic inhibition, a depolarizing voltage step from -70 to 0 mV or a threshold current stimulus adjusted to elicit action potential(s) was applied to a mitral cell using either a nystatin-perforated or conventional whole-cell configuration. We found that an agonist for group II metabotropic glutamate receptors (mGluR2/mGluR3), DCG-IV [(2S,1'R,2'R,3'R)-2-(2,3-dicarboxycyclopropyl)glycine], suppressed, whereas the mGluR2/mGluR3 antagonist LY341495 [(αS)-α-amino-α-[(1S,2S)-2-carboxycyclopropyl]-9H-xanthine-9-propanoic acid] enhanced dendrodendritic inhibition. Genetic ablation of mGluR2 markedly impaired the effects of DCG-IV and LY341495 on dendrodendritic inhibition. DCG-IV reduced both the frequency and the amplitude of spontaneous miniature excitatory postsynaptic currents recorded from granule cells. Additionally, DCG-IV inhibited high-voltage-activated calcium currents in both mitral and granule cells. These results suggest that mGluR2 reduces dendrodendritic inhibition by inhibiting synaptic transmission between mitral cells and granule cells in the AOB.

This review examines the organizational principles underlying olfactory learning in three specialized contexts that occur during sensitive periods of enhanced neural plasticity and emphasizes some of their common features. All three forms of olfactory learning are associated with neural changes in the olfactory bulb (OB) at the first stage of sensory processing. These changes require the association of the olfactory and somatosensory signals in the OB. They all depend on somatosensory stimulation- induced release of noradrenaline that induces structural and functional changes at mitral-granule cell reciprocal synapses in the OB, resulting in increases in inhibitory transmission. In the accessory olfactory bulb, this represents the enhanced self-inhibition of mitral cells, which selectively disrupts the transmission of the mating male's pregnancy-blocking signal at this level. In contrast, an extensive network of secondary dendrites of mitral cells in the main olfactory bulb probably results in a sharpening of the odor-induced pattern of activity, due to increases in lateral inhibition, leading to offspring recognition in sheep and neonatal learning in rats and rabbits. These findings show that inhibitory interneurons play a critical role in olfactory learning. Further work on how these neurons shape olfactory circuit function could provide important clues to understand memory functions of interneurons in other systems. Moreover, recent research has suggested that three forms of olfactory learning are controlled by synergistic, redundant, and distributed neural mechanisms. This has general implications regarding the mechanisms that may contribute to the robustness of memories.

Cholinergic modulation of spontaneous GABAergic currents (mIPSC) was studied using whole-cell patch methods in mouse accessory olfactory bulb slices. Carbachol (above 100 microM) administration produced an increase in the mIPSC frequency in mitral cells, but did not affect the responses of mitral cells to GABA. The carbachol effect persisted in the presence of combined ionotropic and metabotropic glutamatergic receptor antagonists. The carbachol effect was reduced by the muscarinic receptor type-1 and -4 (M1 and M4) antagonist pirenzepine (10 microM), but not by the M2 and M4 antagonist himbacine (10 microM). The KCNQ/Kv7 potassium channel openers retigabine (80 microM) and diclofenac (300 microM) blocked the carbachol action, while the KCNQ potassium channel blocker XE-911 (20 microM) increased the mIPSC frequency. XE-911's action persisted in the presence of glutamate receptor blockers. In the presence of carbachol, mIPSCs were abolished by Ni (200 microM), while being insensitive to the calcium channel blocker nimodipine (30 microM), suggesting a role for R-type calcium channels in the GABA release. These results suggest that carbachol closed KCNQ channels by stimulating M1 receptors on granule cell dendrites, and the resulting depolarized and unstable membrane promoted calcium influx, thus increasing the GABA release. The possible role of acetylcholine in facilitating formation of a pheromone memory in mice is also discussed.

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.

This study examined the effect of suckling on nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d, a histochemical marker for nitric oxide synthase, NOS) reactivity and neuronal NOS mRNA expression in the paraventricular (PVN) and supraoptic (SON) nuclei of lactating rats. Freely nursing (non-separated) dams and those separated from pups for 12 h and then reunited for 0, 15, 30, 60, 90, 120 and 180 min were used for the study. Dams separated from pups and sacrificed at time zero (without reunion) showed a significant decrease in NADPH-d staining and NADPH-d positive cells as well as in the NOS mRNA expression in the PVN and SON compared to that observed in non-separated dams. Reunion with pups and restoration of suckling significantly increased NADPH-d reactivity after 15, 30, 60 min, but not after 90, 120 and 180 min compared to non-reunited pups-deprived dams. A pattern of NADPH-d reactivity and neuronal NOS mRNA expression indistinguishable from that observed during free lactation was reinstated shortly (15 min) after the restoration of suckling stimulus, suggesting that the NADPH-d reactivity in lactation depends on the presence of the suckling stimulus. These results show that suckling stimulus may play a modulatory role in the regulation of NOS reactivity in the magnocellular neurones of the hypothalamic PVN and SON during lactation.

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.

When female mice are mated, they form a memory to the pheromonal signal of their male partner. The neural changes underlying this memory occur in the accessory olfactory bulb, depend upon vaginocervical stimulation at mating and involve changes at the reciprocal synapses between mitral and granule cells. However, the action of vaginocervical stimulation on the reciprocal interactions between mitral and granule cells remains to be elucidated. We have examined the effects of vaginocervical stimulation on paired-pulse depression of amygdala-evoked field potentials recorded in the external plexiform layer of the accessory olfactory bulb (AOB) and the single-unit activity of mitral cells antidromically stimulated from the amygdala in urethane-anaesthetized female mice. Artificial vaginocervical stimulation reduced paired-pulse depression (considered to be due to feedback inhibition of the mitral cell dendrites from the granule cells via reciprocal dendrodendritic synapses) recorded in the AOB external plexiform layer. As would be expected from this result, vaginocervical stimulation also enhanced the spontaneous activity of a proportion of the mitral cells tested. These results suggest that vaginocervical stimulation reduces dendrodendritic feedback inhibition to mitral cells and enhances their activity.

The mouse accessory olfactory bulb contains a high density of nitric oxide synthase and, in females, is involved in the formation of a mating and pheromone-specific recognition memory. The exact role of nitric oxide in this memory model is not yet clearly understood. In this study, in situ hybridization was used to assess neuronal nitric oxide synthase mRNA expression during the critical interval associated with synaptic plasticity in the accessory olfactory bulb of female mice leading to the formation of a recognition memory for the stud male pheromones present following mating. Nitric oxide synthase mRNA was significantly increased following mating and 120-min stud male exposure compared with oestrus mice. The mRNA expression was more predominant in the anterior than the posterior regions of the bulb. These observations indicate a stimulus-specific activation of nitric oxide gene expression in the female mouse accessory olfactory bulb and support the hypothesis that nitric oxide may modulate intermediate synaptic pathways during the formation of a pheromone-dependent olfactory recognition for stud males.

In this study, we examined the effect of acute capsaicin injection on nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d, a histochemical index of nitric oxide, NO, synthase) and Fos expression in the rat pituitary gland. Compared with vehicle, capsaicin significantly activated Fos expression in the anterior and intermediate lobes. In addition, capsaicin-treated rats showed a significant upregulation of NADPH-d in the anterior and neural lobes. Pretreatment of the animals with a specific NO synthase inhibitor, N ω -nitro- l -arginine methyl ester ( l -NAME), significantly attenuated the capsaicin-induced Fos expression in the anterior and intermediate lobes. These observations suggest that NO is a key regulator of the acute effect of capsaicin on the pituitary gland.

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.

Although capsaicin has been shown to activate certain neuronal groups in the hypothalamus and amygdala, the neurotransmitters involved and the exact mechanism of action are not clearly understood at present. The aim of this study was to examine the hypothesis that the effect of capsaicin in the rat hypothalamus and amygdala primarily involves direct activation of the endogenous nitric oxide synthase (NOS) neurons responsible for the synthesis of nitric oxide (NO). Subcutaneous capsaicin injection in male rats, compared with vehicle, caused a significant increase in Fos expression in the paraventricular nucleus (PVN), supraoptic nucleus (SON), and medial and cortical amygdala. The expression of nicotinamide adenine dinucleotide phosphate diaphorase, a histochemical marker for NOS, was also increased in these brain areas in addition to the periventricular and lateral hypothalamic area and central amygdaloid nucleus. Also, capsaicin significantly increased the expression of neuronal NOS messenger RNA and protein in the PVN, SON, and medial amygdala as demonstrated by in situ hybridization and immunohistochemistry, respectively. A higher proportion of the NOS neurons in the PVN, periventricular region, SON and amygdala showed Fos expression in response to capsaicin than vehicle injection. There was little, if any, Fos activation in the NOS-positive neurons in the lateral hypothalamic area. The capsaicin-induced activation of the hypothalamic PVN and SON neurons and the medial amygdaloid nucleus was attenuated in the NOS inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME) -pretreated animals in comparison with the inactive enantiomer D-NAME. These observations indicate that activation of the endogenous NOS system and production of NO constitute a major pathway through which capsaicin exerts its effect within the hypothalamus and amygdala.

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.

Amygdala (AMYG) stimulation caused marked increase in the circulating level of glucagon and these stimulatory effects were abolished by lesions of stria terminalis (ST). Stimulation of dorsal hippocampus (DHPC), on the other hand, greatly decreased the glucagon level and lesions of dorsal fornix (DFX) abolished these stimulatory effects. These results suggested that the reciprocal influence of AMYG and DHPC on plasma level of glucagon played some role in the regulation of glucagon release and these limbic structure had hypothalamic-mediated effects on glucagon release.

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.

This study examined a possible functional involvement of nitric oxide (NO) in the median eminence (ME) and arcuate nucleus (ARC) after capsaicin treatment in rats. Subcutaneous injection of capsaicin increased nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) activity in the ARC-ME compared with vehicle treatment. Fos expression was increased in the ARC after capsaicin injection compared with vehicle-treated rats. Pretreatment with the NO synthase (NOS) inhibitor, N(omega)-nitro-L-arginine methyl ester (L-NAME) attenuated the effect of capsaicin on Fos expression and NADPH-d reactivity in the ARC-ME in comparison with rats injected with D-NAME, the inactive stereoisomer of L-NAME. These observations suggest that NO makes a major contribution to the response of the ARC-ME to a stressor such as capsaicin.

The present report describes neurosteroid modulation of olfactory bulb function by examining the effects of intrabulbar infusion of dehydroepiandrosterone sulfate (DHEAS), a neurohormone discovered in rat brain, on field potentials in the granule cell layer evoked by paired-pulse stimulation of the mouse lateral olfactory tract. Infusion of DHEAS (5 nmol) significantly decreased the test response without affecting the conditioning response. As a consequence, DHEAS selectively potentiated paired-pulse depression, which is believed to be due to granule cell-mediated inhibition of the mitral/tufted cells. The granule-to-mitral/tufted dendrodendritic synapse is GABAergic. Taken together, these results suggest that DHEAS potentiates the GABAergic dendrodendritic inhibition exerted by the granule cells on the mitral/tufted cells.

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.

Attachment to an abusive caregiver has wide phylogenetic representation, suggesting that animal models are useful in understanding the neural basis underlying this phenomenon and subsequent behavioral outcomes. We previously developed a rat model, in which we use classical conditioning to parallel learning processes evoked during secure attachment (odor-stroke, with stroke mimicking tactile stimulation from the caregiver) or attachment despite adversity (odor-shock, with shock mimicking maltreatment). Here we extend this model to mice. We conditioned infant mice (postnatal day (PN) 7–9 or 13–14) with presentations of peppermint odor and either stroking or shock. We used 14C 2-deoxyglucose (2-DG) to assess olfactory bulb and amygdala metabolic changes following learning. PN7-9 mice learned to prefer an odor following either odor-stroke or shock conditioning, whereas odor-shock conditioning at PN13-14 resulted in aversion/fear learning. 2-DG data indicated enhanced bulbar activity in PN7-9 preference learning, whereas significant amygdala activity was present following aversion learning at PN13-14. Overall, the mouse results parallel behavioral and neural results in the rat model of attachment, and provide the foundation for the use of transgenic and knockout models to assess the impact of both genetic (biological vulnerabilities) and environmental factors (abusive) on attachment-related behaviors and behavioral development.

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.

To investigate the effects of Eriobotrya japonica seed extract (ESE) on cellular aging, intracellular calcium homeostasis in young and senescent cells was analyzed using a rat fibroblast culture as an in vitro model system and a calcium imaging technique. The application of bradykinin (BK) transiently elicited intracellular calcium ion (Ca(2+)) increased in most of the young fibroblasts, whereas these responses were scarcely observed or were significantly attenuated in senescent cells. However, the long-term treatment of senescent cells with ESE (for 7 days) dose-dependently increased the amplitude of BK-induced responses and the percentage of BK-responding cells. In particular, most senescent cells could respond to BK with long-term treatment with ESE (1.0% or 2.0%), an effect that reinstated the percentage of BK-responding cells to the same level as that in young cells. The effects of ESE on amplitude or percentage of responding cells were not observed in young cells. Moreover, the time to half decay, which was significantly longer in senescent cells than that in young cells, was shortened in senescent cells with long-term treatment with ESE. These results suggest that treatment with an adequate concentration of ESE renders BK-induced Ca(2+) dynamics in senescent cells similar to those in young cells. Therefore, ESE can retard and/or protect against cellular aging and may be useful for elucidating the antiaging processes.

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.

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.

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.

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 previously established a primary culture system of the accessory olfactory bulb (AOB) to investigate the functional roles of individual types of neuron in pheromonal signal processing. However, the detailed characteristics of cultured AOB neurons were not yet apparent. In the present study, we address the cytological aspects of cultured AOB neurons using immunocytochemical staining methods. Cultured AOB neurons were compared with cultured main olfactory bulb (MOB) neurons in neuronal composition, maturational time course, and cell size. The number of total neurons, measured by microtubule-associated protein 2 (MAP2) immunostaining, progressively decreased, and glutamic acid decarboxylase positive (GAD+) interneurons were scarcely changed in their number in both AOB and MOB cultures over the culture periods. In contrast, the number of tyrosine hydroxylase positive (TH+) neurons in AOB cultures showed a slight, but significant, increase over time in culture, while those in MOB cultures remarkably decreased. The numbers of total neurons and GAD+ neurons were significantly greater in AOB cultures than in MOB cultures at all investigated time points. However, the numbers of TH+ neurons were lower at 7 days in vitro (DIV) and greater at 21 DIV in AOB cultures than in MOB cultures. The somatic sizes of all types of neurons at 14 DIV were significantly larger in AOB cultures than in MOB cultures. Furthermore, the frequency distributions of somatic sizes of total, GAD+, and TH+ neurons were significantly different between AOB and MOB cultures. These subtle differences in vitro may reflect in vivo differences between the AOB and MOB.

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.

Although oxytocin (OT) within the olfactory bulb has been implicated in maternal behaviour and olfactory recognition, the cellular mechanisms of action remain to be clarified. We examined the effects of OT on glutamatergic spontaneous excitatory postsynaptic currents (sEPSCs) in cultured granule cells with the use of whole-cell patch-clamp recordings. OT reversibly increased both the frequency and amplitude of sEPSCs. The effects of OT on sEPSCs were blocked by the selective OT receptor antagonist desGly-NH(2)(9),d (CH(2))(5)-[Thy(Me)(2),Thr(4)]-ornithine vasotocin. OT had no detectable effect, however, on high voltage-activated Ca2+ currents in mitral/tufted cells, suggesting that OT acts presynaptically on step(s) in the release process downstream from calcium influx. OT augmented the membrane current in granule cells evoked by exogenous application of glutamate, indicating a postsynaptic site of action. These results indicate that OT facilitates sEPSCs in granule cells by both pre- and postsynaptic mechanisms.

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.