Neuroprotective effect of oxytocin on cognitive dysfunction, DNA damage, and intracellular chloride disturbance in young mice after cranial irradiation.

Cranial radiation therapy (CRT) is an effective treatment for brain tumors; however, it also causes brain injuries. The pediatric brain is considered especially vulnerable compared to the adult brain; thus, brain injuries caused by CRT may severely affect their quality of life. In this study, we determined the neuroprotective effects of nasal oxytocin administration following cranial radiation in mice. We investigated the cognitive behavior of mice (novel object recognition test and novel object location test), phosphorylated histone H2AX (γ-H2AX) and K+-Cl- transporter (KCC2) by immunohistochemical analysis of the hippocampal sections, and neuronal cells by immunocytochemistry after radiation and oxytocin administration. We found that the number of γ-H2AX foci was increased, and the surface signal intensity of KCC2 immunofluorescence was decreased in cells that were irradiated with X-rays (1.5 Gy, for three consecutive days) compared with cells that were not. Furthermore, using MQAE, we found that the intracellular chloride ion concentration was downregulated in oxytocin-treated cells by increasing surface KCC2 expression. These results indicate that nasal oxytocin administration after cranial irradiation attenuates cognitive dysfunction in mice and exerts multifaceted neuroprotective effects on DNA damage and maintains chloride ion concentration in neuronal cells.

Dorsal telencephalon-specific Nprl2- and Nprl3-knockout mice: novel mouse models for GATORopathy.

The most frequent genetic cause of focal epilepsies is variations in the GAP activity toward RAGs 1 complex genes DEP domain containing 5 (DEPDC5), nitrogen permease regulator 2-like protein (NPRL2) and nitrogen permease regulator 3-like protein (NPRL3). Because these variations are frequent and associated with a broad spectrum of focal epilepsies, a unique pathology categorized as GATORopathy can be conceptualized. Animal models recapitulating the clinical features of patients are essential to decipher GATORopathy. Although several genetically modified animal models recapitulate DEPDC5-related epilepsy, no models have been reported for NPRL2- or NPRL3-related epilepsies. Here, we conditionally deleted Nprl2 and Nprl3 from the dorsal telencephalon in mice [Emx1cre/+; Nprl2f/f (Nprl2-cKO) and Emx1cre/+; Nprl3f/f (Nprl3-cKO)] and compared their phenotypes with Nprl2+/-, Nprl3+/- and Emx1cre/+; Depdc5f/f (Depdc5-cKO) mice. Nprl2-cKO and Nprl3-cKO mice recapitulated the major abnormal features of patients-spontaneous seizures, and dysmorphic enlarged neuronal cells with increased mechanistic target of rapamycin complex 1 signaling-similar to Depdc5-cKO mice. Chronic postnatal rapamycin administration dramatically prolonged the survival period and inhibited seizure occurrence but not enlarged neuronal cells in Nprl2-cKO and Nprl3-cKO mice. However, the benefit of rapamycin after withdrawal was less durable in Nprl2- and Nprl3-cKO mice compared with Depdc5-cKO mice. Further studies using these conditional knockout mice will be useful for understanding GATORopathy and for the identification of novel therapeutic targets.

Stem cell spreading dynamics intrinsically differentiate acral melanomas from nevi.

Early differential diagnosis between malignant and benign tumors and their underlying intrinsic differences are the most critical issues for life-threatening cancers. To study whether human acral melanomas, deadly cancers that occur on non-hair-bearing skin, have distinct origins that underlie their invasive capability, we develop fate-tracing technologies of melanocyte stem cells in sweat glands (glandular McSCs) and in melanoma models in mice and compare the cellular dynamics with human melanoma. Herein, we report that glandular McSCs self-renew to expand their migratory progeny in response to genotoxic stress and trauma to generate invasive melanomas in mice that mimic human acral melanomas. The analysis of melanocytic lesions in human volar skin reveals that genetically unstable McSCs expand in sweat glands and in the surrounding epidermis in melanomas but not in nevi. The detection of such cell spreading dynamics provides an innovative method for an early differential diagnosis of acral melanomas from nevi.

Preservation of vision after CaMKII-mediated protection of retinal ganglion cells.

Retinal ganglion cells (RGCs) are the sole output neurons that transmit visual information from the retina to the brain. Diverse insults and pathological states cause degeneration of RGC somas and axons leading to irreversible vision loss. A fundamental question is whether manipulation of a key regulator of RGC survival can protect RGCs from diverse insults and pathological states, and ultimately preserve vision. Here, we report that CaMKII-CREB signaling is compromised after excitotoxic injury to RGC somas or optic nerve injury to RGC axons, and reactivation of this pathway robustly protects RGCs from both injuries. CaMKII activity also promotes RGC survival in the normal retina. Further, reactivation of CaMKII protects RGCs in two glaucoma models where RGCs degenerate from elevated intraocular pressure or genetic deficiency. Last, CaMKII reactivation protects long-distance RGC axon projections in vivo and preserves visual function, from the retina to the visual cortex, and visually guided behavior.

Dopaminergic Signaling in the Nucleus Accumbens Modulates Stress-Coping Strategies during Inescapable Stress.

Maladaptation to stress is a critical risk factor in stress-related disorders, such as major depression and post-traumatic stress disorder (PTSD). Dopamine signaling in the nucleus accumbens (NAc) has been shown to modulate behavior by reinforcing learning and evading aversive stimuli, which are important for the survival of animals under environmental challenges such as stress. However, the mechanisms through which dopaminergic transmission responds to stressful events and subsequently regulates its downstream neuronal activity during stress remain unknown. To investigate how dopamine signaling modulates stress-coping behavior, we measured the subsecond fluctuation of extracellular dopamine concentration and pH using fast scanning cyclic voltammetry (FSCV) in the NAc, a postsynaptic target of midbrain dopaminergic neurons, in male mice engaged in a tail suspension test (TST). The results revealed a transient decrease in dopamine concentration and an increase in pH levels when the animals changed behaviors, from being immobile to struggling. Interestingly, optogenetic inhibition of dopamine release in NAc, potentiated the struggling behavior in animals under the TST. We then addressed the causal relationship of such a dopaminergic transmission with behavioral alterations by knocking out both the dopamine receptors, i.e., D1 and D2, in the NAc using viral vector-mediated genome editing. Behavioral analyses revealed that male D1 knock-out mice showed significantly more struggling bouts and longer struggling durations during the TST, while male D2 knock-out mice did not. Our results therefore indicate that D1 dopaminergic signaling in the NAc plays a pivotal role in the modulation of stress-coping behaviors in animals under tail suspension stress.SIGNIFICANCE STATEMENT The tail suspension test (TST) has been widely used as a despair-based behavioral assessment to screen the antidepressant so long. Despite its prevalence in the animal studies, the neural substrate underlying the changes of behavior during the test remains unclear. This study provides an evidence for a role of dopaminergic transmission in the modulation of stress-coping behavior during the TST, a despair test widely used to screen the antidepressants in rodents. Taking into consideration the fact that the dopamine metabolism is upregulated by almost all antidepressants, a part of which acts directly on the dopaminergic transmission, current results would uncover the molecular mechanism through which the dopaminergic signaling mediates antidepressant effect with facilitation of the recovery from the despair-like behavior in the TST.

Elevated H2 O2 levels in trinitrobenzene sulfate-induced colitis rats contributes to visceral hyperalgesia through interaction with the transient receptor potential ankyrin 1 cation channel.

BACKGROUND AND AIM: Inflammatory bowel disease is associated with chronic abdominal pain. Transient receptor potential ankyrin 1 (TRPA1) is a well-known pain sensor expressed in primary sensory neurons. Recent studies indicate that reactive oxygen species such as hydrogen peroxide (H2 O2 ) may activate TRPA1. METHODS: Colonic inflammation was induced by intra-colonic administration of trinitrobenzene sulfate (TNBS) in adult male Sprague-Dawley rats. Visceromotor response (VMR) to colorectal distention (CRD) was recorded to evaluate the visceral hyperalgesia. Rats were sacrificed 1 day after treatment with saline or TNBS; colonic tissues from the inflamed region were removed and then processed to assess the H2 O2 content. H2 O2 scavenger N-acetyl-l-cysteine or a TRPA1 antagonist, HC-030031, was intravenously administrated to the TNBS-treated rats or saline-treated rats. In a parallel experiment, intra-colonic H2 O2 -induced visceral hyperalgesia in naïve rats and the effect of intravenous HC-030031 were measured based on the VMR to CRD. RESULTS: Trinitrobenzene sulfate treatment resulted in significant increase in VMR to CRD at day 1. The H2 O2 content in the inflamed region of the colon in TNBS-treated rats was significantly higher than that of saline-treated rats. N-acetyl-l-cysteine or HC-030031 significantly suppressed the enhanced VMR in TNBS-treated rats while saline-treated rats remained unaffected. Moreover, blockade of TRPA1 activation by HC-030031 significantly reversed the exogenous H2 O2 -induced visceral hyperalgesia. CONCLUSION: These results suggest that H2 O2 content of the colonic tissue is increased in the early stage of TNBS-induced colitis. The increased H2 O2 content may contribute to the visceral hyperalgesia by activating TRPA1.

Neuronal Heterotopias Affect the Activities of Distant Brain Areas and Lead to Behavioral Deficits.

Neuronal heterotopia refers to brain malformations resulting from deficits of neuronal migration. Individuals with heterotopias show a high incidence of neurological deficits, such as epilepsy. More recently, it has come to be recognized that focal heterotopias may also show a range of psychiatric problems, including cognitive and behavioral impairments. However, because focal heterotopias are not always located in the brain areas responsible for the symptoms, the causal relationship between the symptoms and heterotopias remains elusive. In this study, we showed that mice with focal heterotopias in the somatosensory cortex generated by in utero electroporation exhibited spatial working memory deficit and low competitive dominance behavior, which have been shown to be closely associated with the activity of the medial prefrontal cortex (mPFC) in rodents. Analysis of the mPFC activity revealed that the immediate-early gene expression was decreased and the local field potentials of the mPFC were altered in the mice with heterotopias compared with the control mice. Moreover, activation of these ectopic and overlying sister neurons using the DREADD (designer receptor exclusively activated by designer drug) system improved the working memory deficits. These findings suggest that cortical regions containing focal heterotopias can affect distant brain regions and give rise to behavioral abnormalities. Significance statement: Recent studies reported that patients with heterotopias have a variety of clinical symptoms, such as cognitive disturbance, psychiatric symptoms, and autistic behavior. However, the causal relationship between the symptoms and heterotopias remains elusive. Here we showed that mice with focal heterotopias in the somatosensory cortex generated by in utero electroporation exhibited behavioral deficits that have been shown to be associated with the mPFC activity in rodents. The existence of heterotopias indeed altered the neural activities of the mPFC, and direct manipulation of the neural activity of the ectopic neurons and their sister neurons in the overlying cortex improved the behavioral deficit. Thus, our results indicate that focal heterotopias could affect the activities of distant brain areas and cause behavioral abnormalities.

A genetically female brain is required for a regular reproductive cycle in chicken brain chimeras.

Sexual differentiation leads to structural and behavioural differences between males and females. Here we investigate the intrinsic sex identity of the brain by constructing chicken chimeras in which the brain primordium is switched between male and female identities before gonadal development. We find that the female chimeras with male brains display delayed sexual maturation and irregular oviposition cycles, although their behaviour, plasma concentrations of sex steroids and luteinizing hormone levels are normal. The male chimeras with female brains show phenotypes similar to typical cocks. In the perinatal period, oestrogen concentrations in the genetically male brain are higher than those in the genetically female brain. Our study demonstrates that male brain cells retain male sex identity and do not differentiate into female cells to drive the normal oestrous cycle, even when situated in the female hormonal milieu. This is clear evidence for a sex-specific feature that develops independent of gonadal steroids.

A potential neuroprotective role of apolipoprotein E-containing lipoproteins through low density lipoprotein receptor-related protein 1 in normal tension glaucoma.

Glaucoma is an optic neuropathy and the second major cause of blindness worldwide next to cataracts. The protection from retinal ganglion cell (RGC) loss, one of the main characteristics of glaucoma, would be a straightforward treatment for this disorder. However, the clinical application of neuroprotection has not, so far, been successful. Here, we report that apolipoprotein E-containing lipoproteins (E-LPs) protect primary cultured RGCs from Ca(2+)-dependent, and mitochondrion-mediated, apoptosis induced by glutamate. Binding of E-LPs to the low density lipoprotein receptor-related protein 1 recruited the N-methyl-d-aspartate receptor, blocked intracellular Ca(2+) elevation, and inactivated glycogen synthase kinase 3ß, thereby inhibiting apoptosis. When compared with contralateral eyes treated with phosphate-buffered saline, intravitreal administration of E-LPs protected against RGC loss in glutamate aspartate transporter-deficient mice, a model of normal tension glaucoma that causes glaucomatous optic neuropathy without elevation of intraocular pressure. Although the presence of α2-macroglobulin, another ligand of the low density lipoprotein receptor-related protein 1, interfered with the neuroprotective effect of E-LPs against glutamate-induced neurotoxicity, the addition of E-LPs overcame the inhibitory effect of α2-macroglobulin. These findings may provide a potential therapeutic strategy for normal tension glaucoma by an LRP1-mediated pathway.

Pleiotropic role for MYCN in medulloblastoma.

Medulloblastoma (MB) is the most common malignant brain tumor of childhood. Sonic Hedgehog (SHH) signaling drives a minority of MB, correlating with desmoplastic pathology and favorable outcome. The majority, however, arises independently of SHH and displays classic or large cell anaplastic (LCA) pathology and poor prognosis. To identify common signaling abnormalities, we profiled mRNA, demonstrating misexpression of MYCN in the majority of human MB and negligible expression in normal cerebella. We clarified a role in pathogenesis by targeting MYCN (and luciferase) to cerebella of transgenic mice. MYCN-driven MB showed either classic or LCA pathologies, with Shh signaling activated in approximately 5% of tumors, demonstrating that MYCN can drive MB independently of Shh. MB arose at high penetrance, consistent with a role for MYCN in initiation. Tumor burden correlated with bioluminescence, with rare metastatic spread to the leptomeninges, suggesting roles for MYCN in both progression and metastasis. Transient pharmacological down-regulation of MYCN led to both clearance and senescence of tumor cells, and improved survival. Targeted expression of MYCN thus contributes to initiation, progression, and maintenance of MB, suggesting a central role for MYCN in pathogenesis.

Maternal microchimerism in underlying pathogenesis of biliary atresia: quantification and phenotypes of maternal cells in the liver.

OBJECTIVE: The goal was to examine whether microchimerism plays a crucial role in the pathogenesis of biliary atresia; we analyzed the localization of maternal microchimeric cells and their phenotypes. METHODS: Liver biopsy specimens from 8 male infants with biliary atresia and 6 control subjects with other liver diseases were investigated for maternal chimeric cells and their phenotypes through double-staining fluorescence in situ hybridization and immunohistochemical analyses. RESULTS: Significantly larger numbers of maternal XX+ cells were found in the portal area and sinusoids of patients with biliary atresia, in comparison with control patients. In phenotypic analyses of XX+ cells, CD8+ T cells, CD45+ cells, and cytokeratin-positive cells were found, and the numbers and proportions among total CD8+ T cells were significantly higher than those in control patients. CONCLUSIONS: Significantly more maternal chimeric CD8+ T cells in the livers of patients with biliary atresia suggest that maternal immunologic insults represent the underlying pathogenesis in biliary atresia. The findings support the recently postulated mechanisms of alloautoimmune and/or autoalloimmune responses.

Activation of cholecystokinin neurons in the dorsal pallium of the telencephalon is indispensable for the acquisition of chick imprinting behavior.

Chick imprinting behavior is a good model for the study of learning and memory. Imprinting object is recognized and processed in the visual wulst, and the memory is stored in the intermediate medial mesopallium in the dorsal pallium of the telencephalon. We identified chicken cholecystokinin (CCK)-expressing cells localized in these area. The number of CCK mRNA-positive cells increased in chicks underwent imprinting training, and these cells expressed nuclear Fos immunoreactivity at high frequency in these regions. Most of these CCK-positive cells were glutamatergic and negative for parvalbumin immunoreactivity. Semi-quantitative PCR analysis revealed that the CCK mRNA levels were significantly increased in the trained chicks compared with untrained chicks. In contrast, the increase in CCK- and c-Fos-double-positive cells associated with the training was not observed after closure of the critical period. These results indicate that CCK cells in the dorsal pallium are activated acutely by visual training that can elicit imprinting. In addition, the CCK receptor antagonist significantly suppressed the acquisition of memory. These results suggest that the activation of CCK cells in the visual wulst as well as in the intermediate medial mesopallium by visual stimuli is indispensable for the acquisition of visual imprinting.

The potential role of glutamate transporters in the pathogenesis of normal tension glaucoma.

Glaucoma, a progressive optic neuropathy due to retinal ganglion cell (RGC) degeneration, is one of the leading causes of irreversible blindness. Although glaucoma is often associated with elevated intraocular pressure (IOP), IOP elevation is not detected in a significant subset of glaucomas, such as normal tension glaucoma (NTG). Moreover, in some glaucoma patients, significant IOP reduction does not prevent progression of the disease. Thus, understanding IOP-independent mechanisms of RGC loss is important. Here, we show that mice deficient in the glutamate transporters GLAST or EAAC1 demonstrate spontaneous RGC and optic nerve degeneration without elevated IOP. In GLAST-deficient mice, the glutathione level in Müller glia was decreased; administration of glutamate receptor blocker prevented RGC loss. In EAAC1-deficient mice, RGCs were more vulnerable to oxidative stress. These findings suggest that glutamate transporters are necessary both to prevent excitotoxic retinal damage and to synthesize glutathione, a major cellular antioxidant and tripeptide of glutamate, cysteine, and glycine. We believe these mice are the first animal models of NTG that offer a powerful system for investigating mechanisms of neurodegeneration in NTG and developing therapies directed at IOP-independent mechanisms of RGC loss.

Hydrogen peroxide-induced thymidine incorporation into cultured rat astrocytes.

We characterized [methyl-(3)H]thymidine ([(3)H]thymidine) and [5-(3)H]uridine ([(3)H]uridine) incorporation into cultured astrocytes and neurons in the presence and absence of hydrogen peroxide (H2O2) in order to define the response to oxidative stress in the central nervous system. [(3)H]Thymidine incorporation into cultured astrocytes was remarkably decreased by N(6),2'-O-dibutyryladenosine 3',5'-cyclic monophosphate (DBcAMP), a permeable analogue of cAMP, which induced a morphological change from the polygonal form (undifferentiated astrocytes) to the process-bearing one (differentiated astrocytes). H2O2 induced [(3)H]thymidine, but not [(3)H]uridine, incorporation into cultured astrocytes at only an early time from 24 h after DBcAMP treatment, although the absolute quantities of [(3)H]thymidine incorporation into astrocytes pretreated with DBcAMP were less than those into astrocytes pretreated without DBcAMP. Hydroxyurea, a replicative DNA synthesis inhibitor, suppressed dose-dependently and completely [(3)H]thymidine incorporation into astrocytes pretreated without DBcAMP, but not astrocytes pretreated with DBcAMP. H2O2 did not stimulate [(3)H]thymidine or [(3)H]uridine incorporation into astrocytes pretreated without DBcAMP and neurons. These findings indicate that only astrocytes pretreated with DBcAMP are able to increase thymidine incorporation specifically in the presence of H2O2 for a purpose other than proliferation, including the repair of H2O2-induced DNA injury, for example.

Unique anti-apoptotic activity of EAAC1 in injured motor neurons.

Injured motor neurons of the adult rat can survive, whereas similar axotomy causes gradual motor neuron death in the adult mouse. We report that the decreased expression of the neuronal glutamate transporter excitatory amino-acid carrier 1 (EAAC1) following nerve injury is associated with motor neuron death in the mouse. Glutamate transporters play a crucial role in prevention of neuronal death by suppressing glutamate toxicity. However, the possible functional role of EAAC1 in preventing neuron death has not been resolved as compared with glial glutamate transporters such as GLT-1. Here, we have revealed a unique 'rescue' function of EAAC1, which is independent of removal of extracellular glutamate. During apoptotic stimuli, a mitochondrial protein, holocytochrome c synthetase (HCCS), translocates to outside the mitochondria, binds to and suppresses the X-linked inhibitor of apoptosis protein (XIAP), leading to activation of caspase-3. The N-terminus of EAAC1 can bind to HCCS, which interferes with the HCCS-XIAP association, and thereby maintain XIAP activity. This unique anti-apoptotic mechanism of EAAC1 functions in rescuing PC12 cells and motor neurons from NGF deprivation and nerve injury, respectively.

Effect of p75NTR on the regulation of naturally occurring cell death and retinal ganglion cell number in the mouse eye.

Neurotrophins induce neural cell survival and differentiation during retinal development and regeneration through the high-affinity tyrosine kinase (Trk) receptors. On the other hand, nerve growth factor (NGF) binding to the low-affinity neurotrophin receptor p75 (p75(NTR)) might induce programmed cell death (PCD) in the early phase of retinal development. In the present study, we examined the retinal cell types that experience p75(NTR)-induced PCD and identify them to be postmitotic retinal ganglion cells (RGCs). However, retinal morphology, RGC number, and BrdU-positive cell number in p75(NTR) knockout (KO) mouse were normal after embryonic day 15 (E15). In chick retina, migratory RGCs express p75(NTR), whereas layered RGCs express the high-affinity NGF receptor TrkA, which may switch the pro-apoptotic signaling of p75(NTR) into a neurotrophic one. In contrast to the chick model, migratory RGCs express TrkA, while stratified RGCs express p75(NTR) in mouse retina. However, RGC number in TrkA KO mouse was also normal at birth. We next examined the expression of transforming growth factor beta (TGFbeta) receptor, which modulates chick RGC number in combination with p75(NTR), but was absent in mouse RGCs. p75(NTR) and TrkA seem to be involved in the regulation of mouse RGC number in the early phase of retinal development, but the number may be later adjusted by other molecules. These results suggest the different mechanism of RGC number control between mouse and chick retina.

Glutamate transport by retinal Muller cells in glutamate/aspartate transporter-knockout mice.

Glutamate transporters are involved in maintaining extracellular glutamate at a low level to ensure a high signal-to-noise ratio for glutamatergic neurotransmission and to protect neurons from excitotoxic damage. The mammalian retina is known to express the excitatory amino acid transporters, EAAT1-5; however, their specific role in glutamate homeostasis is poorly understood. To examine the role of the glial glutamate/aspartate transporter (GLAST) in the retina, we have studied glutamate transport by Muller cells in GLAST-/- mice, using biochemical, electrophysiological, and immunocytochemical techniques. Glutamate uptake assays indicated that the Km value for glutamate uptake was similar in wild-type and GLAST-/- mouse retinas, but the Vmax was approximately 50% lower in the mutant. In Na+-free medium, the Vmax was further reduced by 40%. In patch-clamp recordings of dissociated Muller cells from GLAST-/- mice, application of 0.1 mM glutamate evoked no current showing that the cells lacked functional electrogenic glutamate transporters. The result also indicated that there was no compensatory upregulation of EAATs in Muller cells. [3H]D-Aspartate uptake autoradiography, however, showed that Na+-dependent, high-affinity transporters account for most of the glutamate uptake by Muller cells, and that Na+-independent glutamate transport is negligible. Additional experiments showed that the residual glutamate uptake in Muller cells in the GLAST-/- mouse retina is not due to known glutamate transporters-cystine-glutamate exchanger, ASCT-1, AGT-1, or other heteroexchangers. The present study shows that while several known glutamate transporters are expressed by mammalian Muller cells, new Na+-dependent, high-affinity glutamate transporters remain to be identified.

Effects of estradiol and progesterone on radial maze performance in middle-aged female rats fed a low-calcium diet.

There is increasing evidence that ovarian steroids and calcium ions are involved in learning and memory. To examine the effect of ovarian steroids on learning and memory under a low-calcium condition, middle-aged female rats were fed either a low-calcium (0.02% Ca) or a normal-calcium (1.25% Ca) diet. All rats were ovariectomized (OVX), and these animals were divided into eight groups: 1) an OVX group with a normal-calcium diet (OVX-normal-Ca group), 2) an OVX group with 17beta-estradiol treatment and a normal-calcium diet (E2 group), 3) an OVX with progesterone treatment and a normal-calcium diet (P4 group), 4) an OVX with 17beta-estradiol and progesterone treatments and a normal-calcium diet (E2 + P4 group), 5) an OVX group with a low-calcium diet (OVX-low-Ca group), 6) an OVX group with 17beta-estradiol treatment and a low-calcium diet (LE2 group), 7) an OVX group with progesterone treatment and a low-calcium diet (LP4 group), and 8) an OVX group with 17beta-estradiol and progesterone treatments and a low-calcium diet (LE2 + LP4). Seventy-seven days after the OVX operation, the learning and memory abilities of the rats were examined by using an eight-arm radial maze task. E2 and E2 + P4 groups learned in fewer trials, and performed better in the radial maze and the working memory task than the other groups under the normal-calcium condition. Rats in the LE2 group learned in fewer trials, and performed better in the maze and working memory task than the other low-calcium groups, but in combination with progesterone under the low-calcium condition (LE2 + LP4 group), the facilitative effect of estradiol in all the tasks was inhibited. Treatment with progesterone alone did not inhibit the learning and memory task performance. These results suggest the possibility that treatment with estradiol under low-calcium conditions cannot improve impaired learning and memory when progesterone is applied simultaneously, and that the intake of adequate calcium may be necessary and effective for patients with learning and memory hypofunction receiving hormone replacement therapy.

Inhibitory effects of group II mGluR-related drugs on memory performance in mice.

The cAMP/protein kinase A signaling pathway is negatively modulated by group II metabotropic glutamate receptors (mGluRs), and the cross-talk that occurs between these receptors may modulate learning and memory. To examine the relationship among cAMP/PKA-signaling pathway activity, group II mGluRs, and learning and memory, mice were trained to perform a step-through-type passive avoidance task, and 10 min before each avoidance trial the following drugs were injected intracisternally (i.cist.): vehicle (0.05% dimethylsulfoxide); a specific group II mGluR agonist, DCG-IV (1-50 ng/mouse); a specific group II mGluR antagonist, LY341495 (10-300 ng); a selective inhibitor of cAMP-specific phosphodiesterase, rolipram (100-1000 ng); an activator of adenylyl cyclase, forskolin (25-250 ng); a specific inhibitor of PKA, H-89 (150 or 300 ng) or; an activator of protein kinase C, phorbol 12-myristate 13-acetate (PMA 200 ng). DCG-IV (25 and 50 ng) or LY341495 (150 and 300 ng) reduced the latency in the avoidance task. The reduction of latency by DCG-IV was not observed in mice coinjected with DCG-IV (50 ng) together with rolipram (500 ng) or forskolin (25 ng). Conversely, coinjection of LY341495 with 100 or 1000 ng rolipram, or with 25 or 250 ng forskolin tended to potentiate the LY341495-induced shortening of latency. In addition, the reduction of latency by DCG-IV (50 ng) was not observed in mice coinjected with DCG-IV and PMA together. However, the reduction of latency by LY341495 (300 ng) was potentiated when the drug was coadministered with PMA. These results suggest that changes in the cAMP/PKA-signaling pathway, mediated by group II mGluRs, influence memory in the passive avoidance task, and that both the excessive activation and deactivation of this pathway may induce the impairment of learning and memory.

Diverse NF-kappaB expression in epiretinal membranes after human diabetic retinopathy and proliferative vitreoretinopathy.

PURPOSE: Formation of epiretinal membranes (ERMs) after proliferative diabetic retinopathy (PDR) and proliferative vitreoretinopathy (PVR) results in progressive deterioration of vision, but its pathogenic mechanisms are still unknown. This study was conducted to examine the role of nuclear factor kappa B (NF-kappaB) in the formation of ERMs after PDR and PVR. METHODS: ERM samples were obtained by vitrectomy from 10 patients with PDR (aged 53+/-12 years with 14+/-5 years of diabetes), 20 patients with PVR, and 17 patients with idiopathic ERMs. Ten PVR and 17 idiopathic ERM samples were processed for reverse transcription-polymerase chain reaction (RT-PCR) analysis. In addition, 10 PDR and 10 PVR membranes were processed for immunohistochemical analysis. RESULTS: NF-kappaB mRNA expression levels were significantly higher (10 of 10 versus 9 of 17 subjects in idiopathic ERM, p=0.0119) in PVR subjects. Immunohistochemical analysis showed NF-kappaB protein expression in 8 of the 10 PDR samples as well as all 10 PVR samples, and NF-kappaB positive cells were partially double labeled with glial cell markers. Interestingly, NF-kappaB protein was also overlapped with angiogenic factor interleukin-8 (IL-8) in glial cells as well as vascular endothelial cells. CONCLUSIONS: These results suggest that NF-kappaB is involved in the formation of both glial and vascular endothelial cell components, and that these two cell types might have functional interactions that lead to the enlargement of intraocular proliferative membranes.