Food-finding Test Research Articles

Food Finding Test without Deprivation: A Sensorial Paradigm Sensitive to Sex, Genotype, and Isolation Shows Signatures of Derangements in Old Mice with Alzheimer's Disease Pathology and Normal Aging.

The Food Finding Test (FFT) olfactory paradigm without overnight food deprivation examined olfaction in aged (16-months-old) animals. Ethograms of three goal-directed behaviors towards hidden food (sniffing, finding and eating) elicited in male and female 3xTg-AD mice for Alzheimer's disease (AD) and their age-matched C57BL/6 wild-type counterparts with normal aging were meticulously analyzed with the support of video recordings. The new FFT protocol elicited longer ethograms than previously reported with the standard deprivation protocol. However, it was sensitive when identifying genotype- and sex-dependent olfactory signatures for the temporal patterns of slow sniffing, finding, and eating in AD and males, but it had a striking consistency in females. The impact of forced social isolation was studied and it was found to exert sex-dependent modifications of the ethogram, mostly in males. Still, in both sexes, a functional derangement was detected since the internal correlations among the behaviors decreased or were lost under isolated conditions. In conclusion, the new paradigm without overnight deprivation was sensitive to sex (males), genotype (AD), and social context (isolation-dependent changes) in its ethogram and functional correlation. At the translational level, it is a warning about the impact of isolation in the advanced stages of the disease, paying notable attention to the male sex.

Polarization to M1-type microglia in the hippocampus is involved in depression-like behavior in a mouse model of olfactory dysfunction

Impaired olfactory function may be associated with the development of psychiatric disorders such as depression and anxiety; however, knowledge on the mechanisms underlying psychiatric disorders is incomplete. A reversible model of olfactory dysfunction, zinc sulfate (ZnSO4) nasal-treated mice, exhibit depression-like behavior accompanying olfactory dysfunction. Therefore, we investigated olfactory function and depression-like behaviors in ZnSO4-treated mice using the buried food finding test and tail suspension test, respectively; investigated the changes in the hippocampal microglial activity and neurogenesis in the dentate gyrus by immunohistochemistry; and evaluated the inflammation and microglial polarity related-proteins in the hippocampus using western blot study. On day 14 after treatment, ZnSO4-treated mice showed depression-like behavior in the tail suspension test and recovery of the olfactory function in the buried food finding test. In the hippocampus of ZnSO4-treated mice, expression levels of ionized calcium-binding adapter molecule 1 (Iba1), cluster of differentiation 40, inducible nitric oxide synthase, interleukin (IL)-1β, IL-6, tumor necrosis factor-α, cleaved caspase-3, as well as the number of Iba1-positive cells and cell body size increased, and arginase-1 expression and neurogenesis decreased. Except for the increased IL-6, these changes were prevented by a microglia activation inhibitor, minocycline. The findings suggest that neuroinflammation due to polarization of M1-type hippocampal microglia is involved in depression accompanied with olfactory dysfunction.

Donepezil prevents olfactory dysfunction and α-synuclein aggregation in the olfactory bulb by enhancing autophagy in zinc sulfate-treated mice

Alzheimer’s disease is associated with marked olfactory dysfunction observed in the early stages. Clinical studies reported that acetylcholinesterase inhibitor donepezil (DNP) attenuated this deficit; however, the underlying mechanism remains unclear. Herein, we aimed to examine the effects and underlying mechanisms of DNP on olfactory deficits in zinc sulfate (ZnSO4) nasal-treated mice, which were used as a model of reversible olfactory impairment. We evaluated olfactory function using the buried food finding test and neurogenesis in the subventricular zone (SVZ) using immunohistochemistry. Finally, we measured the expression of doublecortin (DCX), neuronal nuclear antigen (NeuN), olfactory marker protein, tyrosine hydroxylase (TH), tryptophan hydroxylase 2, glutamic acid decarboxylase 67, p-α-synuclein (Ser129), α-synuclein, p-AMPK, p-p70S6 kinase (p70S6K) (Thr389), LC3 Ⅱ/Ⅰ, and p-p62 in the olfactory bulb (OB) by western blotting. On day 7 after treatment, ZnSO4-treated mice exhibited prolonged time to find the buried food, cell proliferation enhancement in the SVZ, increased NeuN, p-α-synuclein (Ser129), and α-synuclein levels, and decreased DCX and TH levels in the OB; except for TH, these changes normalized on day 14 after treatment. Repeated administration of DNP prevented the ZnSO4-induced changes on day 7 after treatment. Moreover, DNP increased p-AMPK and LC3 Ⅱ/Ⅰ, and decreased p-p70S6K and p-p62 (Ser351) levels in the OB, suggesting that DNP enhances autophagy in the OB. These findings indicate that DNP may help prevent olfactory dysfunction by autophagy that reduces α-synuclein aggregation via the AMPK/mTOC1 pathway.

Olfactory Signatures in the Food Finding Test in Mice With Normal and Alzheimer's Disease-Pathological Aging With Special Concerns on the Effects of Social Isolation.

The temporal course and the severity of the involution of sensory systems through aging can be critical since they ensure the ability to perceive and recognize the world. In older people, sensory impairments significantly increase their risk of biological, psychological, and social impoverishment. Besides this, olfactory loss is considered an early biomarker in Alzheimer’s disease (AD) neurodegenerative process. Here we studied olfactory ethograms in middle-aged male and female gold-standard C57BL/6 mice and 3xTg-AD mice, a genetic model of AD that presents cognitive dysfunction and a conspicuous neuropsychiatric-like phenotype. A paradigm involving 1-day food deprivation was used to investigate the ethological patterns shown in the olfactory inspection of a new cage and the sniffing, finding, and eating of hidden food pellets. The sniffing–find–eat temporal patterns were independent of the loss of weight and unveiled (fast) olfactory signatures in Alzheimer’s disease, differing from those (slow progressive) in normal aging. Male 3xTg-AD mice exhibited an early signature than female mice, opposite to animals with normal aging. The sequence of actions was correlated in male and female 3xTg-AD mice in contrast to control mice. Social isolation, naturally occurring in male 3xTg-AD due to the death of cage mates, emphasized their olfactory patterns and disrupted the behavioral correlates. The paradigm provided distinct contextual, sex, and genotype olfactory ethogram signatures useful to investigate olfactory function in normal and AD-pathological aging. Isolation had an impact on enhancing the changes in the olfactory signature here described, for the first time, in the 3xTg-AD model of Alzheimer’s disease.

Effects of Urban Particulate Matter on the Olfactory System in a Mouse Model.

Exposure to urban particulate matter (UPM) is linked to the aggravation of various health problems. Although the nasal cavity is the first barrier to encounter UPM, there is a lack of studies on the impact of UPM on the olfactory area. The purpose of this study was to investigate the cytotoxic effects of UPM on mouse olfactory epithelium, the underlying pathophysiology involved, and changes in cytokine levels. Mice were divided into 4 groups: control, 400UPM (administered 400 µg UPM daily; standard reference material 1649b; average particle diameter 10.5 μm) 1week, 400UPM 2weeks, and recovery 1week after 400UPM 2weeks (n = 10, 6, 6, and 6, respectively). Olfactory function was evaluated by conducting a food-finding test once a week. The olfactory neuroepithelium was harvested for histologic examination, gene ontology, quantitative real-time polymerase chain reaction, and western blotting. Compared to those in the control group, olfactory marker protein, olfactory receptor 1507, adenylyl cyclase 3, and GNAL mRNA levels were lower and S-100, 2',3'-cyclic nucleotide 30-phosphodiesterase, nerve growth factor receptor-associated protein, brain-derived neurotrophic factor, and tachykinin receptor mRNA levels were higher in the 400UPM group olfactory neuroepithelium. There were no significant differences in neuroepithelial inflammatory marker levels between the 400UPM and saline group. UPM decreased olfactory function and might have cytotoxic effects on the olfactory epithelium. Olfactory ensheathing cells and trigeminal nerve might be related to the regeneration of the olfactory epithelium after olfactory destruction associated with UPM.

Differences in Mechanisms of Steroid Therapy and Olfactory Training for Olfactory Loss in Mice.

Steroid therapy and olfactory training are common treatments for olfactory loss. Systemic steroid treatment is the most effective approach for treating sinonasal olfactory loss. Olfactory training is typically effective for treating sensorineural olfactory loss. However, the differences in mechanisms of steroid therapy and olfactory training for olfactory dysfunction are unclear. The aim of this study was thus to evaluate the differences in mechanisms of olfactory training and steroid therapy. Mice in each group were administered 3-methylindole at a dose of 300 mg/kg. Olfactory function was evaluated with a food-finding test once a week. The olfactory neuroepithelium was harvested for histologic examination and protein analysis. Subsequently, data analysis, gene ontology and pathway analysis, quantitative real-time polymerase chain reaction of mRNA, and western blot analysis were conducted. Mice were divided into four groups according to treatment. Control, anosmia, training, and steroid groups resumed food-finding. MMP27, CCL22 and IL18rap mRNA expression were significantly increased in the training group compared to that in the steroid group. IL1R2 mRNA expression was significantly higher in the olfactory neuroepithelium of steroid-treated mice than in that of the training group mice. Steroid therapy improved olfactory function via anti-inflammatory effects, unlike olfactory training which involved cell regeneration and tissue remodeling. Protein and gene analyses revealed that steroid therapy and olfactory training are underpinned by distinct mechanisms. Selection of the most appropriate treatment will be dependent on the cause of olfactory loss.

Blunt olfaction in sexually sluggish male rats.

Among the intact male rats, a subpopulation has been found to show little or no sexual behavior, even after experiencing several mating sessions. This study investigated whether sexually sluggish (SS) males show behavioral differences from normal copulatory (NC) males, other than those concerning sexual behavior. The olfactory preference of males was measured through the time spent displaying nose-poking behavior directed at sexually active males and estrous females for odor exploration in a three-chamber apparatus. Both the NC and SS males showed a significant preference for the odor of estrous females compared with that of male odors. However, SS males spent significantly less time nose-poking estrous females than NC males. The food-finding test was performed after overnight fasting. Our findings showed that all the NC males found the buried pellet within 5 min, whereas over 60% of the SS males failed to find it. The males were also tested for their ability to find a buried bag containing soiled bedding from estrous female cages. The bag was found by 80% of NC males, but only by 20% of SS males. Our results suggest that SS and NC male rats differ not only in sexual behavior but also in other functions such as olfaction.

Olfactory Ensheathing Cells Mediate Neuroplastic Mechanisms After Olfactory Training in Mouse Model.

Background Several studies have reported beneficial effects of olfactory training (OT) on the olfactory nervous system. However, the mechanisms underlying the regeneration of the olfactory system induced by OT are still under investigation. Objectives To determine the key mechanisms involved in the olfactory system recovery and to assess the neuroplastic effects of OT. Methods Thirty healthy female C57BL/6 mice were randomly allocated to 4 groups: control, n = 6; anosmia (no treatment), n = 8; OT, n = 8; and steroid treatment; n = 8. Except for the control group, mice were administered 3-methylindole. Anosmia was assessed using a food-finding test (FFT). The olfactory neuroepithelium was for histological examinations, gene ontology with pathway analyses, RNA, and protein studies. Results FFT was significantly reduced at 3 weeks in the OT mice versus steroids (78.27 s vs 156.83 s, P < .008) and controls (78.27 s vs 13.14 s, P < .003), although final outcome in the FFT was similar in these groups. Expression of olfactory and neurogenesis marker was higher in the olfactory neuroepithelium of the OT group than in the anosmia group without treatment. The mechanisms underlying olfactory regeneration might be related to early olfactory receptor stimulation, followed by neurotrophic factor stimulation of neuronal plasticity. Conclusion OT can improve olfactory function and accelerate olfactory recovery. The mechanisms underlying olfactory regeneration might be related to an initial stimulation of olfactory receptors followed by neurogenesis. Olfactory ensheathing cells might play an important role in olfactory regeneration following OT, based on the observed changes in messenger ribonucleic acid (mRNA) and protein expression, as well as the findings of the gene analysis.

Human umbilical cord blood plasma alleviates age-related olfactory dysfunction by attenuating peripheral TNF-α expression

Social requirements are needed for living in an aging society and individual longevity. Among them, improved health and medical cares, appropriate for an aging society are strongly demanded. Human cord blood-derived plasma (hUCP) has recently emerged for its unique anti-aging effects. In this study, we investigated brain rejuvenation, particularly olfactory function, that could be achieved by a systemic administration of young blood and its underlying mechanisms. Older than 24-month-old mice were used as an aged group and administered with intravenous injection of hUCP repetitively, eight times. Anti-aging effect of hUCP on olfactory function was evaluated by buried food finding test. To investigate the mode of action of hUCP, brain, serum and spleen of mice were collected for further ex vivo analyses. Systemic injection of hUCP improved aging-associated olfactory deficits, reducing time for finding food. In the brain, although an infiltration of activated microglia and its expression of cathepsin S remarkably decreased, significant changes of proinflammatory factors were not detected. Conversely, peripheral immune balance distinctly switched from predominance of Type 1 helper T (Th1) cells to alternative regulatory T cells (Tregs). These findings indicate that systemic administration of hUCP attenuates age-related neuroinflammation and subsequent olfactory dysfunction by modulating peripheral immune balance toward Treg cells, suggesting another therapeutic function and mechanism of hUCP administration.

Hungry Cat—A Serious Game for Conveying Spatial Information to the Visually Impaired

Navigation is done through obtaining spatial information from the environment and forming a spatial map about it. The visually impaired rely mainly on orientation and mobility training by a certified specialist to acquire spatial navigation skills. However, it is manpower intensive and costly. This research designed and developed a serious game, Hungry Cat. This game can convey spatial information of virtual rooms to children with visual impairment through game playing. An evaluation with 30 visually impaired participants was conducted by allowing them to explore each virtual room in Hungry Cat. After exploration, the food finding test, which is a game mode available in Hungry Cat, was conducted, followed by the physical wire net test to evaluate their ability in forming the spatial mental maps of the virtual rooms. The positive results of the evaluation obtained demonstrate the ability of Hungry Cat, in conveying spatial information about virtual rooms and aiding the development of spatial mental maps of these rooms through game playing.

The neuroplastic effect of olfactory training to the recovery of olfactory system in mouse model.

BackgroundSeveral studies have reported the benefits of olfactory training (OT) in the olfactory nervous system of mouse models. Therefore, in this study we performed next‐generation sequencing to evaluate the effects of OT on mRNA sequencing in the olfactory area.MethodsMice in each group were administered 300 mg of 3‐methylindole per kilogram of mouse weight. The olfactory function was evaluated by a food‐finding test once a week. The olfactory neuroepithelium was harvested for histologic examination and protein analysis. Subsequently, data analysis, gene ontology and pathway analysis, quantitative real‐time polymerase chain reaction of mRNA, and Western blot analysis were conducted.ResultsMice were divided into 4 groups according to treatment. Control, anosmia, training, and steroid group mice resumed food finding. Olfactory Maker Protein, olfr1507, ADCY3, and GNAL mRNA expression was higher in the olfactory neuroepithelium of OT than anosmia group mice. In total, 26,364 mRNAs were analyzed. Comparison of the results of OT vs anosmia revealed that ADCY8,10, GFAP, NGF, NGFR, GFAP, and BDNF mRNAs were upregulated in the gene ontology.ConclusionOT improved olfactory function, as indicated by the food‐finding test. OT improved the olfactory recovery time to stimulate olfactory nerve regeneration. OT may initially stimulate the olfactory receptor, followed by neurogenesis. Steroid therapy and OT operated under completely different mechanisms in the upregulated gene study. These results indicate that OT may be one of the future modalities for treating olfactory impairment.

The effectiveness of platelet-rich plasma in an anosmia-induced mice model.

In this study, we aimed to functionally and morphologically demonstrate the effectiveness of platelet-rich plasma (PRP) on anosmia in a mouse model of anosmia. Animal study. A total of 16 male mice were included. When selecting the mice, the food-finding test (FFT) was used to make sure that the animals could smell, and anosmia was induced by administration of intraperitoneal 3-methylindole. The mice were randomly divided into two groups of eight (groups A and B). After 1 week, topical PRP was administered to the mice in group A and topical saline was administered to the mice in group B. The FFT was again administered at 7, 14, and 21 days. The mice were sacrificed on day 21, the olfactory neuroepithelium was histopathologically examined, and the epithelial damage scores and epithelial thickness were measured. After topical administration of PRP and saline, the difference in the average FFT values of the groups was statistically significant at 7, 14, and 21 days (P < 0,005). During the histopathological examination, the epithelial damage score was statistically significantly lower in the PRP group (P = 0.001) than in the saline group, and epithelial thickness was statistically significantly greater in the PRP group compared to the saline group (P = 0.003). We showed that PRP administration has a curative effect on olfactory functions in an anosmia-induced mice model. However, there is a need for further research before PRP can be considered for use in patients with anosmia in clinical practice. NA. Laryngoscope, 128:E157-E162, 2018.

Expression of the NMDA receptor subunit GluN3A (NR3A) in the olfactory system and its regulatory role on olfaction in the adult mouse.

Glutamate is an excitatory neurotransmitter in the olfactory system and its N-methyl-D-aspartate-(NMDA) receptor subunits [GluN1 (NR1), GluN2A (NR2A), and GluN2B (NR2B)] are expressed at synapses in the olfactory bulb and olfactory epithelium. Thus, glutamatergic neurons and NMDA receptors play key roles in olfaction. GluN3A (NR3A) is a unique inhibitory subunit in the NMDA receptor complex; however, the expression and functional role of GluN3A in the olfactory bulb and epithelium remain unclear. The present study examined the expression patterns of GluN3A in the olfactory bulb and epithelium and explored its functional role in the olfactory system. Immunohistochemical and Western blot analyses revealed that GluN3A is abundantly expressed in different cellular layers of the olfactory bulb and epithelium of the adult wild type (WT) mice. In littermate GluN3A knockout (GluN3A(-/-); KO) mice, the expression of olfactory marker protein normally found in mature olfactory sensory neurons was significantly reduced in the olfactory bulb and epithelium. A butyl alcohol stimulus increased immediate-early gene c-Fos expression in the olfactory system of WT mice, while this response was absent in GluN3A KO mice. The level of phosphorylated Ca(2+)/calmodulin-dependent kinase II was significantly lower in GluN3A KO mice compared to WT mice. In buried food finding test, GluN3A mice took significantly longer time to find food compared to WT mice. Consistently, impaired odor distinguishing ability was seen in GluN3A KO mice. These findings suggest that GluN3A, expressed in the adult olfactory system, plays a significant regulatory role in olfactory development and functional activity.

Intranasal delivery of bone marrow mesenchymal stem cells improved neurovascular regeneration and rescued neuropsychiatric deficits after neonatal stroke in rats.

Neonatal stroke is a major cause of mortality and long-term morbidity in infants and children. Currently, very limited therapeutic strategies are available to protect the developing brain against ischemic damage and promote brain repairs for pediatric patients. Moreover, children who experienced neonatal stroke often have developmental social behavior problems. Cellular therapy using bone marrow mesenchymal stem cells (BMSCs) has emerged as a regenerative therapy after stroke. In the present investigation, neonatal stroke of postnatal day 7 (P7) rat pups was treated with noninvasive and brain-specific intranasal delivery of BMSCs at 6 h and 3 days after stroke (1 × 10(6)cells/animal). Prior to transplantation, BMSCs were subjected to hypoxic preconditioning to enhance their tolerance and regenerative properties. The effects on regenerative activities and stroke-induced sensorimotor and social behavioral deficits were specifically examined at P24 of juvenile age. The BMSC treatment significantly reduced infarct size and blood-brain barrier disruption, promoted angiogenesis, neurogenesis, neurovascular repair, and improved local cerebral blood flow in the ischemic cortex. BMSC-treated rats showed better sensorimotor and olfactory functional recovery than saline-treated animals, measured by the adhesive removal test and buried food finding test. In social behavioral tests, we observed functional and social behavioral deficits in P24 rats subjected to stroke at P7, while the BMSC treatment significantly improved the performance of stroke animals. Overall, intranasal BMSC transplantation after neonatal stroke shows neuroprotection and great potential as a regenerative therapy to enhance neurovascular regeneration and improve functional recovery observed at the juvenile stage of development.

Effects of Statins on the Recovery of Olfactory Function in a 3-Methylindole–Induced Anosmia Mouse Model

Despite the importance of olfactory function, no effective medications have been identified to treat olfactory disorders. This study was performed to evaluate the functional recovery of olfaction damaged by 3-methylindole (3MI) in a mouse model with hydroxymethylglutaryl-coenzyme A reductase inhibitors (statins). In a randomized placebo-controlled trial, 24 healthy female BALB/c mice (aged 9-10 weeks and weighing 18-20 g each) were randomly allocated to statin-treated or control groups. Olfactory loss was induced by i.p. injections of 3MI. Atorvastatin (10 mg/kg) or normal saline was then administered per os with a gastric tube for 3 weeks. The effects of treatment were evaluated by food-finding tests and Western blot analysis. Both groups showed complete losses of olfactory function 1 week after 3MI injection. Three weeks after 3MI injection, 9 of the 12 mice in the statin-treated group (75%) passed a food-finding test, in which they were able to find the food within 3 minutes, at least two times out of three trials. However, only two mice in the control group (16.6%) passed the food-finding test, and this difference was statistically significant (p = 0.004; chi-square test). The expression level of the olfactory marker protein was also elevated in the statin-treated group (p = 0.030; Wilcoxon rank sum test). Statins are associated with recovery of olfaction after 3MI injection in a mouse model.

Relationship between olfactory function and olfactory neuronal population in C57BL6 mice injected intraperitoneally with 3-methylindole

Objective It is not known how many olfactory receptor neurons should be intact to maintain olfaction in mouse models treated with 3-methylindole. The aim of this study is to investigate the relationship between a simple olfactory test outcome and the olfactory neuronal population. Study Design Mouse model. Setting Animal laboratory of the Seoul National University Bundang Hospital. Subjects and Methods Olfactory dysfunction was induced by intraperitoneal injection of 3-methylindole in 38 six-week-old female C57BL6 mice. Olfactory function was evaluated by a food-finding test following 72-hour starvation. The olfactory neuronal population was quantified by olfactory marker protein (OMP) expression. Results The average time for finding food was 8.1 seconds in control mice. It was 13.4, 84.4, 90.1, and 111.4 seconds for mice injected with 100, 200, 300, and 400 μg/g of 3-methylindole, respectively. Harvesting the whole olfactory neuroepithelium, densitometric analysis showed significant decrease of OMP in the 300- and 400-μg/g groups as compared with controls (18.8% and 17.5% of relative density, respectively). In the olfactory bulb, there was a significant decrease of OMP in the 200-, 300-, and 400-μg/g groups (44.5%, 37.0%, and 9.0% of relative density, respectively). The food-finding time had a significant reverse correlation with the relative density of OMP both in the olfactory bulb and in the olfactory neuroepithelium. Conclusion Our study showed that olfactory impairment was correlated with olfactory neuronal population in mice treated with 3-methylindole. The food-finding test would be a useful tool that could be easily performed without special training in the 3-methylindole-treated C57BL6 anosmic mouse model.

Transplantation of Neural Stem Cells in Anosmic Mice

ObjectivesTreating olfactory dysfunction is a challenge for physicians. One of the therapeutic options could be transplantation of stem cells. In this study, neural stem cells were transplanted into anosmic mice.MethodsNeural stem cells were generated from the olfactory bulb of green fluorescent protein (GFP)-transgenic C57BL6 mice. Anosmia were induced by injection of intraperitoneal 3-methylindole. The neural stem cells were transplanted transnasally on the next day. The olfactory function was evaluated by a food-finding test once a week. The olfactory neuroepithelium was harvested for histologic examination and protein analysis at 4 weeks.ResultsTwenty-five percent (6/24) of the control mice that were not transplanted with neural stem cells survived at 4 weeks while 67% (8/12) of the transplanted mice survived (P=0.029). The food finding test showed that the transplanted mice resumed finding food at 3 weeks while the control mice resumed finding food at 4 weeks. GFP-positive cells were observed in the olfactory neuroepithelium of the transplanted mice. Western blotting revealed that the olfactory marker protein expression was significantly lower in the control mice than that in the transplanted mice.ConclusionThis study demonstrated that improvement of mouse survival was achieved and recovery of olfactory function was promoted by transnasal transplantation of neural stem cells in the anosmic mouse model. These results indicate that stem cells might be one of the future modalities for treating olfactory impairment.

The effect of Ginkgo biloba on the expression of intermediate–early antigen (c-fos) in the experimentally induced anosmic mouse

Objective Treatment of olfactory dysfunction is very difficult and has limited modality. Treatment with steroids has been used in patients with olfactory dysfunction but the side effects of steroid need to be weighed against its potential benefits. In the present study, the effect of systemic administration of dexamethasone and EGb 761 on damage to olfactory mucosa produced by zinc sulfate was examined. Expression of the immediate-early antigen (IEG), c-fos, in the olfactory bulb and piriform cortex was used to determine the effects of treatment. Methods Young adult CD1 mice (6 to 8 weeks old, male) were used. After anosmic mice were made by bilateral intranasal irrigation with 0.2 ml of 5% (0.17 M) zinc sulfate, anosmia was confirmed by a food finding test. Four groups of anosmic mice were studied: a steroid group (steroid injection group, n = 12), an EGb group (EGb injection group, n = 12), a steroid-EGb group (steroid and EGb injection group, n = 12), and a control group (anosmic mice and no Tx. n = 12). The olfactory bulb and piriform cortex of four mice in each group were obtained at 1, 2, and 3 weeks after instillation of zinc sulfate by cardiac perfusion, and immunohistochemical staining for c-fos was also performed to evaluate brain activity. In approximately 10 well-defined glomeruli of the olfactory bulb and in one side of the piriform cortex, c-fos (+) cells were counted. Statistical analyses were performed by Kruskal–Wallis one-way analysis of variance (ANOVA) by rank. Results In all experimental groups, c-fos (+) cells increased in a time-dependent manner. The combination treatment of steroid and EGb was the most effective and the no-treatment group the least effective 1 week later after zinc sulfate irrigation. However, 3 weeks later after zinc sulfate irrigation, there was no statistically significant differences in the number of c-fos positive cells among all 4 groups (3 treatment groups and the control group). Conclusion The combination treatment of EGb and steroid enhanced the regeneration of the olfactory pathway after olfactory mucosal injury by zinc sulfate. Our study suggests that EGb could be an effective treatment option for olfactory dysfunction.

Leptin regulates olfactory-mediated behavior in ob/ ob mice

We have investigated olfactory-mediated pre-ingestive behavior in leptin ( ob/ ob) and leptin receptor ( db/ db) mutant mice compared to age- and gender-matched wild-type (wt) mice. Olfactory-mediated behavior was tested using a buried food paradigm 5 times/day at 2-h intervals for 6 days. Mean food-finding times of ob/ ob and db/ db mice were approximately 10 times shorter than those of wt mice. To test the effect of leptin replacement in ob/ ob mice, leptin (1 or 5 μg/g body weight in sterile saline) or carrier was injected i.p. once daily prior to testing. Mean food finding times in ob/ ob mice injected with carrier or with 1 μg/g leptin were similar and were 2–3 times faster than in wt mice. Mean food finding times in ob/ ob mice injected with 5 μg/g leptin tripled compared to carrier-injected ob/ ob mice and were of the same order of magnitude as those of wt mice, suggesting functional leptin replacement. A 3-factor repeated measures ANOVA demonstrated significant differences between the 6 cohorts ( P = 0.0001), food finding times ( P ≤ 0.0001), and cohort by day interaction ( P ≤ 0.0001). Post hoc tests suggested that the ob/ ob + 5 μg/g leptin cohort performed more like the wt cohort in the food-finding test than like the ob/ ob or ob/ ob + carrier cohort. Potential local sites of leptin production and action were identified with immunohistochemistry and in situ hybridization in epithelial and gland cells of the olfactory and nasal mucosae. Our results strongly suggest that leptin acting through leptin receptors modulates olfactory-mediated pre-ingestive behavior.