Repeated antibiotic drug treatment negatively affects memory function and glutamatergic nervous system of the hippocampus in mice.

The gut microbiota is associated with memory; however, the relationship between dysbiosis-induced memory deficits and hippocampal glutamatergic neurons remains unclear. In our study, a mouse dysbiosis model showed impaired memory-related behavior in the passive avoidance test; decreased expression levels of glutaminase, excitatory amino acid transporter (EAAT)1, EAAT2, vesicular glutamate transporter 2, synaptophysin, brain-derived neurotrophic factor, doublecortin, neuronal nuclear protein, glial fibrillary acidic protein, and S100ß; and decreased phosphorylation of N-methyl-D-aspartate receptor subunit 1, calmodulin-dependent protein kinase II, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor subunit 1, and cAMP response element-binding protein in the hippocampus. This suggests that dysbiosis-induced memory dysfunction is associated with the hippocampal glutamatergic nervous system.

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)-1ß, 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.

Cytoprotective effects of Hangekobokuto against corticosterone-induced cell death in HT22 cells.

The hypothalamic-pituitary-adrenal (HPA) system plays an important role in stress response. Chronic stress is thought to induce neuronal damage and contribute to the pathogenesis of psychiatric disorders by causing dysfunction of the HPA system and promoting the production and release of glucocorticoids, including corticosterone and cortisol. Several clinical studies have demonstrated the efficacy of herbal medicines in treating psychiatric disorders; however, their effects on corticosterone-induced neuronal cell death remain unclear. Here, we used HT22 cells to evaluate the neuroprotective potential of herbal medicines used in neuropsychiatry against corticosterone-induced hippocampal neuronal cell death. Cell death was assessed using 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) reduction and Live/Dead assays. Hangekobokuto, Kamikihito, Saikokaryukotsuboreito, Kamishoyosan, and Yokukansan were supplied in the form of water-extracted dried powders. Exposure of HT22 cells to ≥ 100 µM corticosterone decreased MTT values. Exposure to 500 µM corticosterone alone reduced MTT values to 18%, while exposure to 10 µM Mifepristone (RU486)-a glucocorticoid receptor antagonist-restored values to 36%. Corticosterone-induced cell death was partially suppressed by treatment with RU486. At 100 µg/mL, Hangekobokuto significantly suppressed the decrease in MTT values (15-32%) and increase in the percentage of ethidium homodimer-1-positive dead cells caused by corticosterone exposure (78-36%), indicating an inhibitory effect on cell death. By contrast, Kamikihito, Saikokaryukotsuboreito, Kamishoyosan, and Yokukansan did not affect corticosterone-induced cell death. Therefore, our results suggest that Hangekobokuto may ameliorate the onset and progression of psychiatric disorders by suppressing neurological disorders associated with increased levels of glucocorticoids.

Hippocampal and gut AMPK activation attenuates enterocolitis-like symptoms and co-occurring depressive-like behavior in ulcerative colitis model mice: Involvement of brain-gut autophagy.

Patients with inflammatory bowel disease, including ulcerative colitis (UC) and Crohn's disease, have a high incidence of psychiatric disorders, including depression and anxiety. However, the underlying pathogenic mechanism remains unknown. Dextran sulfate sodium (DSS)-treated mice, a model of UC, exhibit depressive-like behavior and reduced adenosine monophosphate-activated protein kinase (AMPK) activity, which regulates various physiological functions in the brain and gut. However, comprehensive studies on UC pathophysiology with co-occurring depression focused on brain-gut AMPK activity are lacking. Therefore, we aimed to investigate whether resveratrol (RES), an AMPK activator, prevented DSS-induced UC-like symptoms and depressive-like behavior. DSS treatment induced UC-like pathology and depressive-like behavior, as assessed via the tail suspension test. Moreover, western blotting and immunohistochemical studies revealed that DSS increased p-p70S6 kinase (Thr389), p62, tumor necrosis factor-α, interleukin (IL)-1ß, IL-18, NLR family pyrin domain containing 3 (NLRP3), cleaved caspase-1, cleaved Gasdermin-D (GSDMD), and cleaved caspase-3 expression levels in the rectum and hippocampus, and increased CD40, iNOS, and Kelch-like ECH-associated protein 1 expression levels, and the number of Iba1-positive cells in the hippocampus, and decreased p-AMPK and LC3II/I expression levels, and the number of NF-E2-related factor 2 (Nrf2)-positive cells, and reduced neurogenesis in the hippocampus. These changes were reversed by the RES administration. RES also enhanced PGC1α and SOD1 expression in the hippocampus of DSS-treated male mice. Moreover, NLRP3 staining was observed in the neurons and microglia, and cleaved GSDMD staining in neurons in the hippocampus of DSS-treated mice. Notably, RES prevented UC-like pathology and depressive-like behavior and enhancement of autophagy, decreased rectal and hippocampal inflammatory cytokines and inflammasome, and induced the Nrf2-PGC1α-SOD1 pathway in the hippocampus, resulting in neurogenesis in the hippocampal dentate gyrus. Our findings suggest that brain-gut AMPK activation may be an important therapeutic strategy in patients with UC and depression.

Experimental Study: Interleukin-31 Augments Morphine-Induced Antinociceptive Activity and Suppress Tolerance Development in Mice.

Morphine-induced antinociception is partially reduced in interleukin-31 (IL-31) receptor A (IL-31RA)-deficient mice, indicating that IL-31RA is crucial for morphine-induced peripheral antinociception. Herein, we examined the combined effects of IL-31 and morphine on the antinociceptive activity and itch-associated scratching behavior (LLS) in mice and elucidated the regulatory mechanisms. A hot-plate test was used to assess antinociception. LLS was automatically detected and recorded via a computer. IL-31RA mRNA expression was assessed using real-time polymerase chain reaction. Repeated pre-treatment with IL-31 resulted in significant antinociceptive activity. Repeated administration of morphine decreased the morphine-induced antinociceptive activity, LLS counts, and regular dose and inhibited IL-31-induced LLS. These results suggested that the repeated administration of morphine depleted inter-neuronal IL-31RA levels, preventing morphine-induced antinociception. Therefore, IL-31 may be helpful as an adjunct analgesic to morphine. To explore the benefits of IL-31, its influence on morphine-induced antinociceptive tolerance in mice was examined. An IL-31 and morphine combination increased the analgesic action, which increased the expression of DRG neuronal IL-31RA, elucidating the site of peripheral antinociception of morphine. This site may induce exocytosis of IL-31RA in the sensory nervous system. Collectively, the suppressive effect of IL-31 on morphine-induced antinociceptive tolerance may result from IL-31RA supplementation in sensory nerves.

Possible involvement of inflammasomes on the post-stroke cognitive impairment in a mouse model of embolic cerebral infarct.

Post-stroke cognitive impairment (PSCI) of the complications after stroke has been shown to be involved in brain proinflammatory cytokines such as interleukin (IL)-1ß (IL-lß) and IL-18. In the present study, we examined using acetic acid-induced embolic cerebral infarct (ECI) mice whether post-stroke inflammasome activation is involved in the development of PSCI. In behavioral tests, long-term learning and memory assessed using the passive avoidance test were impaired after ECI. On the other hand, the impairment of short-term learning and memory assessed using the Y-maze test was not observed. Furthermore, the phosphorylated α-amino-3hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit glutamate receptor 1 (GluR1) at Ser 831 and Ser 845 protein was found to be significantly decreased in the dorsal hippocampus of ECI mice. In addition, the expression levels of ionized calcium-binding adapter protein 1 (Iba1), glial fibrillary acidic protein (GFAP), apoptosis-associated speck-like protein containing a caspase recruitment domain / target of methylation-induced silencing 1 (ASC/TMS1), Caspase-1, IL-1ß, IL-18 and tumor necrosis factor-α (TNF-α) were significantly increased in the dorsal hippocampus of ECI mice. These results indicate that development of PSCI after embolic cerebral infarction is due to a decrease in AMPA receptor subunit GluR1 at Ser831 and Ser845 through the inflammasome activation pathway in the dorsal hippocampus.

Analyzing the Antinociceptive Effect of Interleukin-31 in Mice.

The theory that an itch inhibits pain has been refuted; however, previous research did not investigate this theory for an interleukin-31 (IL-31)-induced itch. Previously, we have found that morphine-induced antinociception was partially reduced in IL-31 receptor A (IL-31RA)-deficient (IL-31RAKI) mice, indicating that IL-31RA may play an important role in morphine-induced peripheral antinociception. In the present study, we evaluated the effect of IL-31-induced analgesia on a 2,4,6-trinitrochlorobenzene (TNCB)-sensitized mice using a hot-plate test. This test evaluated the antinociceptive activity of morphine and non-steroidal anti-inflammatory drugs (NSAIDs). Repeated pretreatment with IL-31 showed significant antinociceptive action. Furthermore, its combination with morphine, but not with NSAIDs, increased the analgesic action. In contrast, treatment with TNCB and capsaicin decreased antinociception. Moreover, TNCB increased IL-31RA expression in the dorsal root ganglia at 24 h, whereas capsaicin inhibited it. The comparative action of several analgesics on TNCB or capsaicin was evaluated using a hot-plate test, which revealed that the antinociceptive activity was decreased or disappeared in response to capsaicin-induced pain in IL-31RAKI mice. These results indicate that the analgesic action of IL-31 involves the peripheral nervous system, which affects sensory nerves. These results provide a basis for developing novel analgesics using this mechanism.

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.

Brexpiprazole prevents colitis-induced depressive-like behavior through myelination in the prefrontal cortex.

Patients with inflammatory bowel disease (IBD) have higher rates of psychiatric pathology including depression. The dextran sulfate sodium (DSS)-treated mice exhibit IBD- and depressive-like phenotypes. A disturbed intestinal environment causes a decrease in serotonin and abnormal myelination in the brain, along with depressive-like behavior in rodents. However, the involvement of these factors in DSS-induced depressive-like behavior in mice remains unclear. In this study, we examined whether myelin proteins in the prefrontal cortex (PFC) and hippocampi were altered in DSS-treated mice, along with the changes in the serotonergic system in the PFC by western blotting and HPLC. The effects of brexpiprazole (Brx), a serotonin modulator, on DSS-induced depressive-like behavior using the tail-suspension test were evaluated. Subsequently, we investigated Brx's effects on the levels of myelin, nodal proteins, and neurotrophic molecules in the PFC with western blotting, and examined the altered node of Ranvier formation by immunohistochemistry. DSS-treated mice showed a reduction in myelin and nodal proteins, dysfunction of the serotonergic system, and impaired formation of the nodes of Ranvier in the PFC. Brx administration prevented the DSS-induced depressive-like behavior and demyelination in the PFC. However, the Brx-mediated effects were inhibited by the selective 5-HT1A antagonist, WAY100635, or the selective TrkB antagonist, ANA-12. Brx decreased the phosphorylation of ERK, CREB, and TrkB along with the expression of BDNF in the PFC of DSS-treated mice. Moreover, the effects of Brx were blocked by WAY100635. These findings indicated that myelination regulated by the activation of the ERK1/2-CREB-BDNF-TrkB pathway in the PFC may be involved in mediating the antidepressant effects of Brx.

Correlation between the reduction in hippocampal SirT2 expression and depressive-like behaviors and neurological abnormalities in olfactory bulbectomized mice.

Reduced activity of hippocampal silent information regulator protein 2 (SirT2) has been associated with the development of depression caused by disturbances in neuronal and synaptic plasticity. However, changes in the hippocampal SirTs in olfactory bulbectomized (OBX) mice, an animal model of depression, remain unknown. Therefore, this study examined depressive-like behaviors, hippocampal SirTs, synaptic plasticity-associated proteins, and cell proliferation in OBX mice. The OBX mice showed depressive-like behaviors; reduced SirT2, synaptophysin, and PSD95 levels; and reduced cell proliferation in the hippocampus. These data indicate that decreased hippocampal SirT2 may contribute to pathophysiological depression and strongly affect the psychological state.

Capsaicin suppresses interleukin-31-induced itching partially involved in inhibiting the expression of dorsal root ganglion interleukin-31 receptor A in male mice.

To elucidate the mechanisms underlying the antipruritic effect of capsaicin, we investigated how topical application of capsaicin (0.01, 0.1 and 1.0% w/v) affects spontaneous scratching in NC/Nga mice, inerleukin-31 (IL-31) induced in BALB/c mice, and IL-31 receptor A (IL-31RA) and transient receptor potential vanilloid member 1 (TRPV1) mRNA expression in dorsal root ganglia (DRG). Capsaicin concentration-dependently suppressed long-lasting scratching (over 1.0 s, itch-associated scratching) and short-lasting scratching (0.3-1.0 s, locomotor activity) immediately after the application. Total long-lasting scratching and short-lasting scratching counts for 24 h and IL-31RA mRNA expression in the DRG significantly decreased with increasing concentration of capsaicin. Furthermore, 1.0% capsaicin suppressed long-lasting scratching and short-lasting scratching for more than 72 h. At this point, DRG IL-31RAmRNA was significantly decreased, but there was no change in cutaneous IL-31RA and TRPV1 mRNA. Thus capsaicin suppresses long-lasting scratching by inhibiting IL-31RA mRNA expression in the DRG. Next, we examined the effect of capsaicin on IL-31-induced long-lasting scratching in BALB/c mice. Repeated administration of IL-31 (50 µg/kg, subcutaneous) every 12 h for 3 days apparently increased long-lasting scratching counts and IL-31RA mRNA in the DRG. These increases were significantly suppressed by pretreatment with 1.0% capsaicin. TRPV1 mRNA in the DRG was also decreased within 1-24 h after capsaicin application. These results suggest that the strong and prolonged antipruritic action for IL-31-induced itching of capsaicin was caused by desensitization of C-fibers, and, in addition, the long-lasting inhibition of IL-31RA mRNA expression in the DRG.

Possible role of transcriptional regulation of 5-HT1A receptor in the midbrain on unadaptation to stress in mice.

The ability to adapt to stress is an essential defensive function of a living body, and disturbance of this ability in the brain may contribute to the development of affective illness. Previously, we reported that mice exposed to unadaptable restraint stress show emotional abnormality. Moreover, this emotional abnormality was alleviated by chronic treatment with flesinoxan, a serotonin (5-HT)1A receptor agonist. 5-HT1A receptor expression is regulated by several transcription factors such as nuclear deformed epidermal autoregulatory factor (NUDR/Deaf-1) and five prime repressors under dual repression binding protein 1 (Freud-1). The present study was designed to investigate the expression levels of 5-HT1A receptor and its transcription factors in the midbrain and hippocampus of stress-adaptive and -unadaptive mice. Mice were exposed to 14 days of repeated adaptable (1 h/day) or repeated unadaptable (4 h/day) restraint stress, or were left in their home cage (non-stressed groups). In a western blot analysis, a significant increase in the expression levels of 5HT1A receptor protein were observed in the hippocampal membrane fraction in stress-adaptive mice. In contrast, the expression levels of 5-HT1A receptor protein in stress-unadaptive mice were significantly increased in both cytoplasmic and membrane fraction of the midbrain. Furthermore, real-time PCR analysis revealed that, in the midbrain of stress-unadaptive mice, the expression levels of 5-HT1A receptor mRNA and Freud-1 or NUDR mRNA were significantly increased and decreased, respectively. These results suggest that increased expression of 5-HT1A receptor due to decrease in the expression of Freud-1 and NUDR in the midbrain may play a pivotal role in the emotional abnormality of stress-unadaptive mice.

Antidepressant effects of Enterococcus faecalis 2001 through the regulation of prefrontal cortical myelination via the enhancement of CREB/BDNF and NF-κB p65/LIF/STAT3 pathways in olfactory bulbectomized mice.

A therapeutic strategy through the gut-brain axis has been proven to be effective in treatment for depression. In our previous study, we demonstrated that Enterococcus faecalis 2001 (EF-2001) prevents colitis-induced depressive-like behavior through the gut-brain axis in mice. More recently, we found that demyelination in the prefrontal cortex (PFC) was associated with depressive-like behavior in an animal model of major depressive disorder, olfactory bulbectomized (OBX) mice. The present study investigated the effects of EF-2001 on depressive-like behaviors in OBX mice and the underlying molecular mechanisms from the perspective of myelination in the PFC. OBX mice exhibited depressive-like behaviors in the tail-suspension, splash, and sucrose preference tests, and decreased myelin and paranodal proteins along with mature oligodendrocytes in the PFC. These behavioral and biochemical changes were all prevented by treatment with EF-2001. Further, EF-2001 treatment increased brain-derived neurotrophic factor (BDNF) and leukemia inhibitory factor (LIF) in the PFC. Interestingly, an immunohistochemical analysis revealed enhanced phospho (p) -cAMP-responsive element binding protein (CREB) expression in neurons, p-nuclear factor-kappa B (NFκB) p65 (Ser536) expression in astrocytes, and p-signal transducer and activator of transcription 3 (STAT3) (Ty705) expression in mature oligodendrocytes in the PFC of OBX mice. From these results, we suggest that EF-2001 administration prevents depressive-like behaviors by regulating prefrontal cortical myelination via the enhancement of CREB/BDNF and NFκB p65/LIF/STAT3 pathways. Our findings strongly support the idea that a therapeutic strategy involving the gut microbiota may be a promising alternative treatment for alleviating symptoms of depression.

Trichostatin A, a histone deacetylase inhibitor, alleviates the emotional abnormality induced by maladaptation to stress in mice.

Recent reports have implied that aberrant biochemical processes in the brain are frequently accompanied by subtle shifts in the cellular epigenetic profile that might underlie the pathogenic progression of psychiatric disorders. The aim of the present study was to examine the effect of trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, on the emotional abnormality induced by maladaptation to stress in mice. Mice were exposed to repeated restraint stress for 240 min/day for 14 days. We applied dosing schedules. In one schedule, from the 3rd day of stress exposure, mice were treated with TSA (1650 µM/4 µL, i.c.v.) immediately after the daily exposure to restraint stress. In the other schedule, from the 1st day of stress exposure, mice were treated with TSA 2 h before exposure to restraint stress. After the final exposure to restraint stress, the emotionality of mice was evaluated using the hole-board test. Mice that were exposed to restraint stress for 240 min/day for 14 days showed a decrease in head-dipping behavior. This decreased emotionality observed in stress-maladaptive mice was significantly recovered by chronic treatment with TSA 2 h before daily exposure to restraint stress, which confirmed the development of stress adaptation. On the other hand, no such stress adaptation was observed under chronic treatment with TSA immediately after daily stress exposure. A biochemical study showed that tryptophan hydroxylase, the rate-limiting enzyme in serotonin (5-HT) synthesis, was increased in midbrain containing raphe nuclei obtained from stress-adapted mice that were chronically treated with TSA 2 h before daily stress exposure. These findings suggest that an HDAC inhibitor may have a beneficial effect on stress adaptation by affecting 5-HT neural function in the brain and alleviate the emotional abnormality under conditions of excessive stress.

Activation of 5-HT1A receptor reduces abnormal emotionality in stress-maladaptive mice by alleviating decreased myelin protein in the ventral hippocampus.

We previously reported that abnormal emotionality in stress-maladaptive mice was ameliorated by chronic treatment with flesinoxan, a 5-HT1A receptor agonist. Furthermore, the maintenance of hippocampal myelination appeared to contribute to the development of stress adaptation in mice. However, the effects of 5-HT1A receptor activation on myelination under the stress-maladaptive situations and the underlying mechanisms remain unknown. In the present study, we examined using flesinoxan whether activation of 5-HT1A receptor can reduce an abnormal emotional response by acting on oligodendrocytes to preserve myelin proteins in stress-maladaptive mice. Mice were exposed to repeated restraint stress for 4 h/day for 14 days as a stress-maladaptive model. Flesinoxan was given intraperitoneally immediately after the daily exposure to restraint stress. After the final exposure to restraint stress, the emotionality of mice was evaluated by the hole-board test. The expression levels of brain-derived neurotrophic factor (BDNF), phosphorylated-extracellular signal-regulated kinase (p-ERK), phosphorylated-cAMP response element-binding protein (p-CREB), myelin-associated glycoprotein (MAG), myelin basic protein (MBP) and oligodendrocyte transcription factor 2 (olig2) in the hippocampus was assessed by western blotting. Hippocampal oligodendrogenesis were examined by immunohistochemistry. Chronic treatment with flesinoxan suppressed the decrease in head-dipping behaviors in stress-maladaptive mice in the hole-board test. Under this condition, the decreases in MAG and MBP in the hippocampus recovered with increase in BDNF, p-ERK, p-CREB, and olig2. Furthermore, hippocampal oligodendrogenesis in stress-maladaptive mice was promoted by chronic treatment with flesinoxan. These findings suggest that 5-HT1A receptor activation may promote oligodendrogenesis and myelination via an ERK/CREB/BDNF signaling pathway in the hippocampus and reduces abnormal emotionality due to maladaptation to excessive stress.

Effects of Gadolinium Deposition in the Brain on Motor or Behavioral Function: A Mouse Model.

Background Recent studies showing gadolinium deposition in multiple organs have raised concerns about the safety of gadolinium-based contrast agents (GBCAs). Purpose To explore whether gadolinium deposition in brain structures will cause any motor or behavioral alterations. Materials and Methods This study was performed from July 2019 to December 2020. Groups of 17 female BALB/c mice were each repeatedly injected with phosphate-buffered saline (control group, group A), a macrocyclic GBCA (group B), or a linear GBCA (group C) for 8 weeks (5 mmol per kilogram of bodyweight per week for GBCAs). Brain MRI studies were performed every other week to observe the signal intensity change caused by the gadolinium deposition. After the injection period, rotarod performance test, open field test, elevated plus-maze test, light-dark anxiety test, locomotor activity assessment test, passive avoidance memory test, Y-maze test, and forced swimming test were performed to assess the locomotor abilities, anxiety level, and memory. Among-group differences were compared by using one-way or two-way factorial analysis of variance with Tukey post hoc testing or Dunnett post hoc testing. Results Gadolinium deposition in the bilateral deep cerebellar nuclei was confirmed with MRI only in mice injected with a linear GBCA. At 8 weeks, contrast ratio of group C (0.11; 95% CI: 0.10, 0.12) was higher than that of group A (-2.1 × 10-3; 95% CI: -0.011, 7.5 × 10-3; P < .001) and group B (2.7 × 10-4; 95% CI: -8.2 × 10-3, 8.7 × 10-3; P < .001). Behavioral analyses showed that locomotor abilities, anxiety level, and long-term or short-term memory were not different in mice injected with linear or macrocyclic GBCAs. Conclusion No motor or behavioral alterations were observed in mice with brain gadolinium deposition. Also, the findings support the safety of macrocyclic gadolinium-based contrast agents. © RSNA, 2021 Online supplemental material is available for this article. See also the editorial by Chen in this issue.

Disturbance of prefrontal cortical myelination in olfactory bulbectomized mice is associated with depressive-like behavior.

Recent studies have reported that demyelination is associated with the development of depression. Olfactory bulbectomized (OBX) rodents are a useful experimental animal model for depressive disorder. However, little is known about the change in myelination in the brain of OBX mice. To address this question, we observed depressive-like behavior of OBX mice in the tail-suspension test, and determined the quantity of myelin proteins in the prefrontal cortex (PFC), striatum and hippocampus on day 14 or 21 after surgery. The number of nodes of Ranvier paired with the paranodal marker contactin-associated protein (Caspr), as well as the numbers of immature and mature oligodendrocytes in the PFC, were also measured on day 21 after surgery. We examined whether these behavioral and neurochemical changes observed in OBX mice were reversed by chronic administration of imipramine. OBX mice showed depressive-like behavior in the tail-suspension test together with a decrease in the levels of myelin proteins such as myelin basic protein, myelin-associated glycoprotein and cyclicnucleotide phosphodiesterase in the PFC on day 21 after surgery. The number of nodes of Ranvier and mature oligodendrocytes were also decreased in the PFC of OBX mice, while the number of immature oligodendrocytes was increased on day 21 after surgery. However, the number of immature oligodendrocytes in the PFC of OBX mice was decreased on day 35 after surgery. Administration of imipramine (20 mg/kg) for 2 weeks from day 21 after surgery improved OBX-induced depressive-like behavior and abnormal myelination in the PFC. The present findings suggest that the disturbance of myelin function in the PFC may contribute to the pathophysiology of depression, and further support the notion that it plays an important role in the psychological state.

Activation of cholinergic system partially rescues olfactory dysfunction-induced learning and memory deficit in mice.

Deficits in olfaction are associated with neurodegenerative disorders such as Alzheimer's disease. A recent study reported that intranasal zinc sulfate (ZnSO4)-treated mice show olfaction and memory deficits. However, it remains unknown whether olfaction deficit-induced learning and memory impairment is associated with the cholinergic system in the brain. In this study, we evaluated olfactory function by the buried food find test, and learning and memory function by the Y-maze and passive avoidance tests in ZnSO4-treated mice. The expression of choline acetyltransferase (ChAT) protein in the olfactory bulb (OB), prefrontal cortex, hippocampus, and amygdala was assessed by western blotting. Moreover, we observed the effect of the acetylcholinesterase inhibitor physostigmine on ZnSO4-induced learning and memory deficits. We found that intranasal ZnSO4-treated mice exhibited olfactory dysfunction, while this change was recovered on day 14 after treatment. Both short-term and long-term learning and memory were impaired on days 4 and 7 after treatment with ZnSO4, whereas the former, but not the latter, was recovered on day 14 after treatment. A significant correlation was observed between olfactory function and short-term memory, but not long-term memory. Treatment with ZnSO4 decreased the ChAT level in the OB on day 4, and increased and decreased the ChAT levels in the OB and hippocampus on day 7, respectively. Physostigmine improved the ZnSO4-induced deficit in short-term, but not long-term, memory. Taken together, the present results suggest that short-term memory may be closely associated with olfactory function via the cholinergic system.

Leukemia Inhibitory Factor Participates in the Formation of Stress Adaptation via Hippocampal Myelination in Mice.

Leukemia inhibitory factor (LIF) has been shown to be involved in myelination. The present study investigated whether LIF plays a significant role in the formation of stress adaptation using stress-adaptive and -maladaptive mice, and also attempted to confirm whether there was any difference in myelination between these animal models. A single exposure to restraint stress for 1 h induced a decrease in head-dipping behavior in the hole-board test. This stress response was not seen in mice that had been exposed to restraint stress for 1 h/day for 14 days, which is referred to as stress adaptation. In contrast, repeated exposure to restraint stress for 4 h/day for 14 days did not lead to the development of stress adaptation, and was still associated with a decrease in head-dipping behaviors. Under these conditions, LIF protein was found to be increased with astrocyte activation in the hippocampus of stress-adaptive mice, whereas no change was observed in stress-maladaptive mice. On the other hand, major myelin proteins including myelin-associated glycoprotein and myelin basic protein, as well as mature oligodendrocytes, were decreased in the hippocampus of stress-maladaptive, but not stress-adaptive, mice. Furthermore, while the levels of phosphorylated-extracellular signal-regulated kinase (p-ERK) were increased in the hippocampus of stress-adaptive, this change did not occur in stress-maladaptive mice. Taken together, the present findings suggest that LIF, which may be derived from activated astrocytes, plays a critical role in the maintenance of hippocampal myelination via an ERK signaling pathway, and contributes to the development of stress adaptation.

Dopamine D2 receptor supersensitivity in the hypothalamus of olfactory bulbectomized mice.

Olfactory bulbectomy (OBX) in rodents induces neurochemical and behavioral changes similar to those observed in individuals with depressive disorders. Our previous study suggested that OBX alters dopaminergic function in the striatum of mice; however, the effects on dopaminergic function in the hypothalamus is unknown. Therefore, in this study we examined dopaminergic system changes in the hypothalamus after OBX. Mice were administrated either the nonselective dopamine (DA) agonist apomorphine or the selective D2 agonist quinelorane, or pretreated with the selective D1 antagonist SCH23390 in combination with the selective D2 antagonist sulpiride or D3 antagonist SB277011A. Body temperature, which is regulated by the hypothalamic dopaminergic system, was monitored to evaluate changes in the dopaminergic system of the hypothalamus. DA D2 receptor (D2DR), tyrosine hydroxylase (TH), and phosphorylated (p)- DA- and cAMP-regulated phosphoprotein-32 (DARPP-32) levels in the hypothalamus were evaluated by western blotting. OBX mice exhibited significantly enhanced apomorphine-induced or quinelorane-induced hypothermia. The apomorphine-induced hypothermic response was reversed by the administration of sulpiride, but not SCH23390 or SB277011A. Moreover, TH and p-DARPP-32 levels were reduced and D2DR increased in the hypothalamus of OBX mice. These findings revealed that the OBX mice display enhanced DA receptor responsiveness associated with the hypothalamus, which may relate to some of the behavioral and neurochemical alterations reported in this animal model. Identification of changes in the hypothalamic dopaminergic system of OBX mice may provide useful information for the development of novel antidepressant treatments.