Overexpression of Swedish mutant APP in aged astrocytes attenuates excitatory synaptic transmission.

Amyloid precursor protein (APP), a type I transmembrane protein, has different aspects, namely, performs essential physiological functions and produces ß-amyloid peptide (Aß). Overexpression of neuronal APP is responsible for synaptic dysfunction. In the central nervous system, astrocytes - a major glial cell type - have an important role in the regulation of synaptic transmission. Although APP is expressed in astrocytes, it remains unclear whether astrocytic overexpression of mutant APP affects synaptic transmission. In this study, the effect of astrocytic overexpression of a mutant APP on the excitatory synaptic transmission was investigated using coculture system of the transgenic (Tg) cortical astrocytes that express the human APP695 polypeptide with the double mutation K670N + M671L found in a large Swedish family with early onset Alzheimer's disease, and wild-type hippocampal neuron. Significant secretion of Aß 1-40 and 1-42 was observed in cultured cortical astrocytes from the Tg2576 transgenic mouse that genetically overexpresses Swedish mutant APP. Under the condition, Tg astrocytes did not affect excitatory synaptic transmission of cocultured wild-type neurons. However, aged Tg astrocytes cultured for 9 weeks elicited a significant decrease in excitatory synaptic transmission in cocultured neurons. Moreover, a reduction in the number of readily releasable synaptic vesicles accompanied a decrease in the number of excitatory synapses in neurons cocultured with aged Tg astrocytes. These observations indicate that astrocytic expression of the mutant APP is involved in the downregulation of synaptic transmission with age.

Ganglioside GM3 is essential for the structural integrity and function of cochlear hair cells.

GM3 synthase (ST3GAL5) is the first biosynthetic enzyme of a- and b-series gangliosides. Patients with GM3 synthase deficiency suffer severe neurological disability and deafness. Eight children (ages 4.1 ± 2.3 years) homozygous for ST3GAL5 c.694C>T had no detectable GM3 (a-series) or GD3 (b-series) in plasma. Their auditory function was characterized by the absence of middle ear muscle reflexes, distortion product otoacoustic emissions and cochlear microphonics, as well as abnormal auditory brainstem responses and cortical auditory-evoked potentials. In St3gal5(-/-) mice, stereocilia of outer hair cells showed signs of degeneration as early as postnatal Day 3 (P3); thereafter, blebs devoid of actin or tubulin appeared at the region of vestigial kinocilia, suggesting impaired vesicular trafficking. Stereocilia of St3gal5(-/-) inner hair cells were fused by P17, and protein tyrosine phosphatase receptor Q, normally linked to myosin VI at the tapered base of stereocilia, was maldistributed along the cell membrane. B4galnt1(-/-) (GM2 synthase-deficient) mice expressing only GM3 and GD3 gangliosides had normal auditory structure and function. Thus, GM3-dependent membrane microdomains might be essential for the proper organization and maintenance of stereocilia in auditory hair cells.

Filament perforation model for mouse subarachnoid hemorrhage: surgical-technical considerations.

OBJECTIVE: Mouse subarachnoid hemorrhage (SAH) models are becoming increasingly important. We aimed to report and discuss the detailed technical-surgical approach and difficulties associated with the circle of Willis perforation (cWp) model, with reference to the existing literature. METHODS: First, the cWp model was reproduced using ddY mice following scarification at 0 h, Days 1, 2, and 3 after SAH. Second, C57BL/6 mice were subjected to SAH with histological examination on Days 1, 2, and 3. Sham-operated mice were sacrificed on Day 2. Neurological performance, amount of subarachnoid blood, cerebral vasospasm (CVS), and neuronal injury were assessed. Relevant articles found in the MEDLINE database were reviewed. RESULTS: Induction of SAH was successfully reproduced. The volume of subarachnoid blood decreased with time due to resorption. Neurological performance was worse in SAH compared with sham. Signs of CVS could be confirmed on Days 2 and 3, but not Day 1. The cumulative number of microthrombi was significantly higher on Days 2 and 3, but not Day 1. Apoptotic and degenerative neurons were found in the cortex and hippocampal area. Our review of the literature revealed the cWp model to be the most frequently used. The present findings largely confirmed previously published results. However, detailed technical-surgical description and its discussion were sparse, which we provide here. CONCLUSIONS: The current study provides additional useful information characterizing the cWp model. This model may be of first choice at present, as important pathologies can be reproduced and most findings in the literature are based on it.

Allogeneic transplantation of fetal membrane-derived mesenchymal stem cell sheets increases neovascularization and improves cardiac function after myocardial infarction in rats.

BACKGROUND: Mesenchymal stem cell (MSC) transplantation has been pursued as a new method to repair damaged myocardium. We focused on the fetal membrane (FM) as an alternative source to bone marrow (BM)-derived MSCs. In this study, we investigated whether transplantation of allogeneic FM-MSC sheets could attenuate myocardial dysfunction in a rat chronic myocardial infarction (MI) model. METHODS: Sheets of allogeneic FM-MSC or autologous BM-MSC were transplanted into the scarred myocardium 4 weeks after coronary ligation. RESULTS: Four weeks after transplantation, both allogeneic FM-MSC and autologous BM-MSC sheets had significantly improved cardiac function and reduced myocardial fibrosis compared with the untreated MI group. In both MSC sheet-transplanted groups, the peri-infarct regional capillary density was increased. Some engrafted MSCs formed vascular structures and were positive for lectin I and α-smooth muscle actin. The numbers of engrafted cells and differentiated cells were very low after both types of MSC sheet transplantation. CD3 T cells did not increase in the transplantation site, but CD163 M2 macrophages increased in the groups transplanted with allogeneic FM-MSC and autologous BM-MSC. CONCLUSIONS: Transplantation of allogeneic FM-MSC or autologous BM-MSC sheets attenuated myocardial dysfunction in a rat MI model to a similar degree. The engraftment rate of transplanted cells and immune cell infiltration into the transplanted area did not differ between the two types of MSC transplants. M2 macrophage induction has possible involvement in the therapeutic effects of MSC transplantation. Allogeneic FM-MSC sheet transplantation might be a new therapeutic strategy after MI.

Injection of specific amyloid-beta oligomers (beta1₋40:beta1₋42 = 10:1) into rat medial septum impairs memory retention without inducing hippocampal apoptosis.

OBJECTIVES: Because of the well-known neurochemical interactions between the septum and hippocampus during memory processes, we investigated the effect of amyloid-beta (A-beta) injection into the medial septum (MS) on the behavior in Wistar rats. We also assessed whether the observed effects were functional or due to apoptosis. METHODS: Specific A-beta oligomers (beta1-40:beta1-42 = 10:1) were injected into MS for seven consecutive days. Behavior was assessed with the Morris water maze task. RESULTS: Compared with the control group, rats that received A-beta oligomers exhibited significant memory retention impairment (P < 0·05) without apoptosis in the cornu ammonis (CA)1 and CA3 regions of the dorsal hippocampus. DISCUSSION: These data indicate that septal injection of A-beta impairs memory retention, even in the absence of hippocampal apoptosis. This result might bring new insight to spatial memory-related disorders like Alzheimer's disease (AD).

Facilitation of memory impairment and cholinergic disturbance in a mouse model of Alzheimer's disease by mild ischemic burden.

Epidemiological studies suggest that cerebral ischemia may contribute to the onset and progression of Alzheimer's disease (AD). However, the mechanism by which ischemic events trigger the onset and progression of AD is poorly understood. Acetylcholine (ACh) is one of the key factors in memory, and cholinergic disturbance is a primary feature of AD. To clarify whether cholinergic disturbance is implicated in the exacerbation of AD symptoms by cerebral ischemia, memory impairment and hippocampal ACh release were examined in young (4-6 month-old) Tg2576 (Tg) mice, an AD transgenic mouse model, and in age-matched control mice with or without transient cerebral ischemia (bilateral common carotid artery occlusion: 2VO). 2VO induced memory impairment and decreased high-K(+)-evoked ACh release in Tg mice, but not in control mice. There were no differences in memory and ACh release between sham-operated control and Tg mice. Increases in ß-amyloid (Aß) 40 and Aß42 were also observed in 2VO-operated Tg mice compared with sham-operated Tg mice, but no evident amyloid plaques or neuronal loss were found in the hippocampus of these mice. These results suggest that the memory of Tg mice is affected by 2VO, and the memory impairment may be due to cholinergic dysfunction induced by Aß. Our findings support the idea that cerebral hypoperfusion could be a risk factor for AD.

Effects of mood stabilizers on marble-burying behavior in mice: involvement of GABAergic system.

RATIONALE: Obsessive-compulsive disorder (OCD) is characterized by recurrent unwanted thoughts (obsessions), usually accompanied by repetitive behaviors (compulsions) intended to alleviate anxiety. Marble-burying behavior is a pharmacological model for study of OCD. OBJECTIVES: In the present study, we examined the effects of mood stabilizers on marble-burying behavior in mice, as well as the role of GABA receptors in this behavior. METHODS: The effects of treatment with valproate, carbamazepine, lithium carbonate, lamotrigine, muscimol and baclofen on marble-burying behavior in mice were evaluated. RESULTS: Valproate (10, 30 and 100 mg/kg, i.p.) and carbamazepine (30 and 100 mg/kg, p.o.) significantly reduced marble-burying behavior without affecting total locomotor activity in ICR mice. Lamotrigine (30 mg/kg, i.p.) also significantly reduced marble-burying behavior in ddY mice. On the other hand, lithium carbonate (10, 30 and 100 mg/kg, i.p.) reduced total locomotor activity without affecting marble-burying behavior in ddY mice. The selective GABA(A) receptor agonist muscimol (1 mg/kg) significantly reduced marble-burying behavior without affecting total locomotor activity, whereas the selective GABA(B) receptor agonist baclofen (3 mg/kg) reduced total locomotor activity without affecting marble-burying behavior. Moreover, the selective GABA(A) receptor antagonist bicuculline (3 mg/kg) significantly counteracted the decrease in marble-burying induced by the administration of muscimol (1 mg/kg) and valproate (100 mg/kg). CONCLUSIONS: These results suggest that GABAergic mechanism is involved in marble-burying behavior, and that valproate, carbamazepine and lamotrigine reduce marble-burying behavior. Moreover, valproate reduces marble-burying behavior via a GABA(A) receptor-dependent mechanism.

Cholinergic involvement and synaptic dynamin 1 expression in Yokukansan-mediated improvement of spatial memory in a rat model of early Alzheimer's disease.

The aim of this study was to investigate the effect of Yokukansan (YKS) on the impairment of spatial memory and cholinergic involvement in a rat model of early-phase Alzheimer's disease (AD). In this model, rats underwent four-vessel transient cerebral ischemia and then were treated with beta amyloid oligomers injected intracerebroventricularly once daily for 7 days. These animals showed memory impairment in an eight-arm radial maze task without histological evidence of apoptosis but with a decrease in expression of hippocampal dynamin 1, an important factor in synaptic vesicle endocytosis. Oral administration of YKS for 2 weeks significantly increased the number of correct choices and decreased the number of error choices in the eight-arm radial maze task (P < 0.05). Moreover, YKS significantly increased high K⁺-evoked potentiation of acetylcholine (ACh) release (P < 0.05) and significantly increased the expression of dynamin 1 (P < 0.01) in the hippocampus. The ameliorative effect of YKS on spatial memory impairment in our rat model of early-phase AD may be mediated in part by an increase in ACh release and modulation of dynamin 1 expression, leading to improved synaptic function. Future studies will determine whether YKS is similarly useful in the treatment of memory defects in patients diagnosed with early-stage AD.

Ameliorative effects of telmisartan on the inflammatory response and impaired spatial memory in a rat model of Alzheimer's disease incorporating additional cerebrovascular disease factors.

Telmisartan, an angiotensin type 1 receptor blocker, is used in the management of hypertension to control blood pressure. In addition, telmisartan has a partial agonistic effect on peroxisome proliferator activated receptor γ (PPARγ). Recently, the effects of telmisartan on spatial memory or the inflammatory response were monitored in a mouse model of Alzheimer's disease (AD). However, to date, no studies have investigated the ameliorative effects of telmisartan on impaired spatial memory and the inflammatory response in an AD animal model incorporating additional cerebrovascular disease factors. In this study, we examined the effect of telmisartan on spatial memory impairment and the inflammatory response in a rat model of AD incorporating additional cerebrovascular disease factors. Rats were subjected to cerebral ischemia and an intracerebroventricular injection of oligomeric or aggregated amyloid-ß (Aß). Oral administration of telmisartan (0.3, 1, 3 mg/kg/d) seven days after ischemia and Aß treatment resulted in better performance in the eight arm radial maze task in a dose-dependent manner. Telmisartan also reduced tumor necrosis factor α mRNA expression in the hippocampal region of rats with impaired spatial memory. These effects of telmisartan were antagonized by GW9662, an antagonist of PPARγ. These results suggest that telmisartan has ameliorative effects on the impairment of spatial memory in a rat model of AD incorporating additional cerebrovascular disease factors via its anti-inflammatory effect.

Preferential involvement of Na⁺/Ca²âº exchanger type-1 in the brain damage caused by transient focal cerebral ischemia in mice.

The Na(+)/Ca(2+) exchanger (NCX), an ion-transporter located in the plasma membrane of neuronal cells, contributes to intracellular Ca(2+) homeostasis. Within the brain, three isoforms (NCX1, NCX2, and NCX3) are widely distributed. However, it is not clear to what extent these isoforms are involved in ischemic brain damage in mammals. We therefore used genetically altered mice and isoform-selective NCX inhibitors in a model of transient focal ischemia to investigate the role of each NCX isoform in ischemic brain damage. NCX isoform-mutant mice (NCX1(+/-), NCX2(+/-), and NCX3(+/-)) and wild-type mice were subjected to 90min of middle cerebral artery occlusion (MCAO) followed by 24h of reperfusion. One of three NCX inhibitors [SN-6, KB-R7943, or SEA0400 (3 or 10mgkg(-1), i.p.)] was administered to ddY mice at 30min before more prolonged (4-h) MCAO followed by 24h of reperfusion. After transient MCAO reperfusion, the cerebral infarcts in NCX1(+/-) mice, but not those in NCX2(+/-) or NCX3(+/-) mice, were significantly smaller than those in wild-type mice. SN-6 and SEA0400, which are more selective for the NCX1 isoform, significantly reduced the infarct volume at 10mg/kg. In contrast, KB-R7943, which is more selective for NCX3, did not. These results suggest that the NCX1 isoform may act preferentially (vs. the NCX2 and NCX3 isoforms) to exacerbate the cerebral damage caused by ischemic insult in mice, and that NCX1-selective inhibitors warrant investigation as a potential therapeutic agents for stroke.

Long-term culture of astrocytes attenuates the readily releasable pool of synaptic vesicles.

The astrocyte is a major glial cell type of the brain, and plays key roles in the formation, maturation, stabilization and elimination of synapses. Thus, changes in astrocyte condition and age can influence information processing at synapses. However, whether and how aging astrocytes affect synaptic function and maturation have not yet been thoroughly investigated. Here, we show the effects of prolonged culture on the ability of astrocytes to induce synapse formation and to modify synaptic transmission, using cultured autaptic neurons. By 9 weeks in culture, astrocytes derived from the mouse cerebral cortex demonstrated increases in ß-galactosidase activity and glial fibrillary acidic protein (GFAP) expression, both of which are characteristic of aging and glial activation in vitro. Autaptic hippocampal neurons plated on these aging astrocytes showed a smaller amount of evoked release of the excitatory neurotransmitter glutamate, and a lower frequency of miniature release of glutamate, both of which were attributable to a reduction in the pool of readily releasable synaptic vesicles. Other features of synaptogenesis and synaptic transmission were retained, for example the ability to induce structural synapses, the presynaptic release probability, the fraction of functional presynaptic nerve terminals, and the ability to recruit functional AMPA and NMDA glutamate receptors to synapses. Thus the presence of aging astrocytes affects the efficiency of synaptic transmission. Given that the pool of readily releasable vesicles is also small at immature synapses, our results are consistent with astrocytic aging leading to retarded synapse maturation.

Hypothalamic 2-arachidonoylglycerol regulates multistage process of high-fat diet preferences.

BACKGROUND: In this study, we examined alterations in the hypothalamic reward system related to high-fat diet (HFD) preferences. We previously reported that hypothalamic 2-arachidonoylglycerol (2-AG) and glial fibrillary acid protein (GFAP) were increased after conditioning to the rewarding properties of a HFD. Here, we hypothesized that increased 2-AG influences the hypothalamic reward system. METHODS: The conditioned place preference test (CPP test) was used to evaluate HFD preferences. Hypothalamic 2-AG was quantified by gas chromatography-mass spectrometry. The expression of GFAP was examined by immunostaining and western blotting. RESULTS: Consumption of a HFD over either 3 or 7 days increased HFD preferences and transiently increased hypothalamic 2-AG levels. HFD consumption over 14 days similarly increased HFD preferences but elicited a long-lasting increase in hypothalamic 2-AG and GFAP levels. The cannabinoid 1 receptor antagonist O-2050 reduced preferences for HFDs after 3, 7, or 14 days of HFD consumption and reduced expression of GFAP after 14 days of HFD consumption. The astrocyte metabolic inhibitor Fluorocitrate blocked HFD preferences after 14 days of HFD consumption. CONCLUSIONS: High levels of 2-AG appear to induce HFD preferences, and activate hypothalamic astrocytes via the cannabinoid system. We propose that there may be two distinct stages in the development of HFD preferences. The induction stage involves a transient increase in 2-AG, whereas the maintenance stage involves a long lasting increase in 2-AG levels and activation of astrocytes. Accordingly, hypothalamic 2-AG may influence the development of HFD preferences.

N-acetyl-L-cysteine inhibits marble-burying behavior in mice.

In the present study, we examined the effect of N-acetyl-L-cysteine (NAC), a glutamate-modulating agent, on marble-burying behavior in mice. Fluvoxamine (30 mg/kg, p.o.) and mirtazapine (3 mg/kg, i.p.) significantly inhibited marble-burying behavior without affecting locomotor activity. Similarity, NAC (150 mg/kg, i.p.) significantly inhibited marble-burying behavior without affecting locomotor activity. On the other hand, the antioxidant α-tocopherol (10, 30, and 100 mg/kg, p.o.) had no effect on the marble-burying behavior. These results suggest that the glutamatergic system is involved in the marble-burying behavior, and NAC may be useful for treatment of OCD.

Role of 5-hydroxytryptamine2C receptors in marble-burying behavior in mice.

We examined the role of 5-hydroxytryptamine(2C) (5-HT(2C)) receptors in marble-burying behavior in mice. When administered alone, the selective 5-HT(2C) agonist WAY161503 (3 mg/kg) inhibited marble-burying behavior. Moreover, the selective 5-HT(2C) antagonist SB242084 (3 mg/kg) reversed the inhibition of marble-burying behavior by 2,5-dimethoxy-4-iodoamphetamine (DOI) (1 mg/kg) or WAY161503 (3 mg/kg). Similarly, SB242084 (1 mg/kg) reversed the inhibition of marble-burying behavior by fluvoxamine (30 mg/kg) or paroxetine (3 mg/kg). These results suggest that 5-HT(2C) receptors play a role in marble-burying behavior in mice.

CD4 and CD8 T cells require different membrane gangliosides for activation.

Initial events of T-cell activation involve movement of the T-cell receptor into lipid rafts. Gangliosides are major components of lipid rafts. While investigating T-cell activation in ganglioside-deficient mice, we observed that CD4(+) and CD8(+) T cells required different ganglioside subsets for activation. Activation of CD4(+) T cells from GM3 synthase-null mice, deficient in GM3-derived gangliosides, is severely compromised, whereas CD8(+) T-cell activation is normal. Conversely, in cells from GM2/GD2 synthase-null mice, expressing only GM3 and GD3, CD4(+) T-cell activation is normal, whereas CD8(+) T-cell activation is deficient. Supplementing the cells with the corresponding missing gangliosides restores normal activation. GM3 synthase-null mice do not develop experimental asthma. Distinct expression patterns of ganglioside species in CD4(+) T and CD8(+) T cells, perhaps in uniquely functional lipid rafts, define immune functions in each T-cell subset. Control of ganglioside expression would offer a strategy targeting for specific T-cell subpopulations to treat immune diseases.

ADAMTS13 gene deletion enhances plasma high-mobility group box1 elevation and neuroinflammation in brain ischemia-reperfusion injury.

Highly adhesive glycoprotein von Willebrand factor (VWF) multimer induces platelet aggregation and leukocyte tethering or extravasation on the injured vascular wall, contributing to microvascular plugging and inflammation in brain ischemia-reperfusion. A disintegrin and metalloproteinase with thrombospondin type-1 motifs 13 (ADAMTS13) cleaves the VWF multimer strand and reduces its prothrombotic and proinflammatory functions. Although ADAMTS13 deficiency is known to amplify post-ischemic cerebral hypoperfusion, there is no report available on the effect of ADAMTS13 on inflammation after brain ischemia. We investigated if ADAMTS13 deficiency intensifies the increase of extracellular HMGB1, a hallmark of post-stroke inflammation, and exacerbates brain injury after ischemia-reperfusion. ADAMTS13 gene knockout (KO) and wild-type (WT) mice were subjected to 30-min middle cerebral artery occlusion (MCAO) and 23.5-h reperfusion under continuous monitoring of regional cerebral blood flow (rCBF). The infarct volume, plasma high-mobility group box1 (HMGB1) level, and immunoreactivity of the ischemic cerebral cortical tissue (double immunofluorescent labeling) against HMGB1/NeuN (neuron-specific nuclear protein) or HMGB1/MPO (myeloperoxidase) were estimated 24 h after MCAO. ADAMTS13KO mice had larger brain infarcts compared with WT 24 h after MCAO (p < 0.05). The rCBF during reperfusion decreased more in ADAMTS13KO mice. The plasma HMGB1 increased more in ADAMTS13KO mice than in WT after ischemia-reperfusion (p < 0.05). Brain ischemia induced more prominent activation of inflammatory cells co-expressing HMGB1 and MPO and more marked neuronal death in the cortical ischemic penumbra of ADAMTS13KO mice. ADAMTS13 deficiency may enhance systemic and brain inflammation associated with HMGB1 neurotoxicity, and aggravate brain damage in mice after brief focal ischemia. We hypothesize that ADAMTS13 protects brain from ischemia-reperfusion injury by regulating VWF-dependent inflammation as well as microvascular plugging.

Involvement of opioid system in cognitive deficits induced by ∆9-tetrahydrocannabinol in rats.

RATIONALE: Cannabis is a widely used illicit substance. ∆(9)-Tetrahydrocannabinol (THC), the major psychoactive component of cannabis, is known to induce cognitive deficits that closely resemble the impairment observed in schizophrenic patients. We previously reported that THC (6 mg/kg) impairs spatial memory in the eight-arm radial maze, and that this memory disturbance was reversed by the cannabinoid CB(1) receptor antagonist rimonabant (0.1 mg/kg), suggesting that the effect of THC is mediated through cannabinoid CB(1) receptors. OBJECTIVES: The present study was designed to examine the possible involvement of opioid receptors in the THC-induced impairment of spatial memory. METHODS: The effects of treatment with the nonselective opioid receptor antagonist naloxone (0.3 and 1 mg/kg), the µ-opioid receptor antagonist ß-funaltrexamine (0.3 and 1 mg/kg), the δ-opioid receptor antagonist naltrindole (1 and 3 mg/kg), and the κ-opioid receptor antagonist nor-binaltorphimine (0.03 and 0.1 mg/kg) on the impairment of spatial memory induced by THC were evaluated using the eight-arm radial maze. RESULTS: The nonselective opioid receptor antagonist naloxone, the µ-opioid receptor antagonist ß-funaltrexamine, and the κ-opioid receptor antagonist nor-binaltorphimine, but not the δ-opioid receptor antagonist naltrindole, attenuated THC-induced cognitive deficits, suggesting an involvement of µ- and κ-opioid receptors in this behavioral response. CONCLUSIONS: These results demonstrate that the endogenous opioid system is involved in the regulation of the acute short-term and working memory deficits induced by cannabis.

Yokukansan enhances pentobarbital-induced sleep in socially isolated mice: possible involvement of GABA(A)-benzodiazepine receptor complex.

In the present study, we investigated the effect of the Kampo medicine Yokukansan (YKS) on pentobarbital-induced sleep in group-housed and socially isolated mice. Socially isolated mice showed shorter sleeping time than the group-housed mice. YKS (300 mg/kg, p.o.) prolonged the pentobarbital-induced sleeping time in socially isolated mice without affecting pentobarbital sleep in group-housed mice. The prolongation of sleeping time by YKS was reversed by bicuculline (3 mg/kg, i.p.) and flumazenil (3 mg/kg, i.p.), but not WAY100635. These findings suggest that the GABA(A)-benzodiazepine receptor complex, but not 5-HT(1A) receptors, is involved in the reversal effect of YKS on the decrease of pentobarbital sleep by social isolation.