Hippocampal acetylcholine receptor activation-dependent long-term depression in streptozotocin-induced diabetic rats.

Cholinergic innervation of the hippocampus correlates with memory formation. In a well-established animal model of type 1 diabetes mellitus, obtained by injecting young adult rats with streptozotocin (STZ), reductions have been reported in the expression of acetylcholine receptors and choline acetyltransferase. In this study, we showed that long-term synaptic depression (LTD) induced by carbachol (CCh), a nonselective cholinergic receptor agonist, at Schaffer collateral-CA1 synapses in hippocampal slices was significantly weaker in streptozotocin-induced diabetic rats (STZ rats) than in age-matched control rats. No significant change was observed in the paired-pulse ratio between before and 80 min after the application of CCh in control and STZ rats. Moreover, CCh-induced LTD in control and STZ rats was not affected by an NMDA receptor antagonist. Although the application of CCh down-regulated the surface expression of GluA2 in the hippocampus of control rats, but not STZ rats. Therefore, the present results suggest that acetylcholine receptor-mediated LTD in STZ rats requires the internalization of AMPA receptors on the postsynaptic surface and their intracellular effects in the hippocampus.

Involvement of trigeminal axons in nose-to-brain delivery of glucagon-like peptide-2 derivative.

In our previous study, we created a glucagon-like peptide-2 (GLP-2) derivative with the functional sequence PAS-CPP to achieve efficient uptake by the respiratory epithelium and trigeminal nerve. By using octaarginine for cell penetrating peptides (CPP) and FFLIPKG, a reverse sequence of a part of the cathepsin D sequence for the penetration accelerating sequence (PAS), we found that the derivative was taken up by the cells through macropinocytosis and efficiently escaped from the endosomes and exited the cells. Moreover, it showed drug effects by intranasal (in.) administration at the same dose as intracerebroventricular (icv.) administration, which is direct drug administration into the brain. The purpose of this study was to elucidate the cause of the drug effect of in. administered PAS-CPP-GLP-2 at the same dose as that by icv. administration. The present results suggested that although icv. administered PAS-CPP-GLP-2 entered the cerebrospinal fluid, it barely penetrated the perivascular space of the brain, and therefore, only a small amount of the administered dose may have reached the site of action in the brain. In contrast, it was qualitatively suggested that in. administered PAS-CPP-GLP-2 migrates from the trigeminal nerve to the central nervous system via the principal sensory trigeminal nucleus and then through the trigeminal lemniscus. The present results show that nose-to-brain delivery by trigeminal axons, which is assumed to be a transcellular pathway, may be possible. As the drug can be delivered into the nerve, it is expected to be applied not only as a central delivery route but also for the treatment of neurological diseases.

Intracerebroventricular administration of oxytocin and intranasal administration of the oxytocin derivative improve ß-amyloid peptide (25-35)-induced memory impairment in mice.

AIM: We previously reported that oxytocin, a peptide hormone, can reverse the ß-amyloid peptide (25-35) (Aß25-35 )-induced impairments of the murine hippocampal synaptic plasticity. In this study, we examined the effects of oxytocin on the Aß25-35 -induced impairment of cognitive behavior in murine in order to investigate the potential of oxytocin as a clinical treatment tool for Alzheimer's disease (AD). METHODS: The Y-maze and Morris water maze (MWM) tests were performed. Since the intracerebroventricular (ICV) administration is both invasive and impractical, we further utilized intranasal (IN) delivery to the brain. For this purpose, we prepared an oxytocin derivative containing cell-penetrating peptides and a penetration accelerating sequence, which was subsequently used in our IN administration experiments. RESULTS: We herein showed that the ICV administration of oxytocin in mice exerted memory-improving effects on the Aß25-35 -induced amnesia in both the Y-maze and MWM tests. The IN administration of the oxytocin derivative exhibited memory-improving effects in the Y-maze test. Moreover, we acquired evidence that the fluorescein isothiocyanate-labeled oxytocin derivative was distributed throughout the mouse brain following its IN administration. CONCLUSION: Our results suggest that the oxytocin derivative is effective for its IN delivery to the brain and may be particularly useful in the clinical treatment of cognitive impairment, such as that characterizing AD.

Long-term exposure to high glucose induces changes in the expression of AMPA receptor subunits and glutamate transmission in primary cultured cortical neurons.

Hyperglycemia, which occurs under the diabetic conditions, induces serious diabetic complications. Diabetic encephalopathy has been defined as one of the major complications of diabetes, and is characterized by neurochemical and neurodegenerative changes. However, little is known about the effect of long-term exposure to high glucose on neuronal cells. In the present study, we showed that exposure to glutamate (100 mM) for 7 days induced toxicity in primary cortical neurons using the MTT assay. Additionally, high glucose increased the sensitivity of AMPA- or NMDA-induced neurotoxicity, and decreased extracellular glutamate levels in primary cortical neurons. In Western blot analyses, the protein levels of the GluA1 and GluA2 subunits of the AMPA receptor as well as synaptophysin in neurons treated with high glucose were significantly increased compared with the control (25 mM glucose). Therefore, long-term exposure to high glucose induced neuronal death through the disruption of glutamate homeostasis.

Fluorescein Permeability of the Blood-Brain Barrier Is Enhanced in Juvenile- but Not Young Adult-Onset Type 1 Diabetes in Rats.

Clinically, neurological disorders, such as cognitive impairments and dementia, have been reported as diabetic complications, which are remarkable, especially in children with diabetes. The blood-brain barrier (BBB) is a physiologically dynamic regulatory barrier that maintains the consistency of the fluid microenvironment composition of the brain. However, the differences in BBB conditions between children and adults and the contribution of the BBB to the severity of cognitive impairments remain unclear. We generated adult-onset diabetes mellitus (DM) and juvenile-onset diabetes mellitus (JDM) diabetic rat models and investigated BBB functions in these models during the early stages of type 1 diabetes. We performed a BBB permeability assay using sodium fluorescein, a small-molecule fluorescent dye, to evaluate endothelial transport from the blood to the central nervous system. One week after diabetes onset, BBB permeability increased in the hippocampus and striatum of JDM rats, but no changes were observed in the frontal cortex and hypothalamus of JDM rats or for any region of DM rats. The double staining of tight junction proteins and astrocytes revealed no changes in the hippocampus and striatum of JDM rats. These results suggested that the observed increase in BBB permeability during early-stage diabetes onset in JDM rats, which did not depend on the expression of the interendothelial tight junction protein, claudin-5, may affect stylized neural development and cognitive function.

Usefulness of cell-penetrating peptides and penetration accelerating sequence for nose-to-brain delivery of glucagon-like peptide-2.

Neuropeptides are expected as therapeutic drug candidates for central nervous system (CNS) disorders. Intracerebroventricular (i.c.v.) administration of glucagon-like peptide-2 (GLP-2) has an antidepressant-like effect not only in depression model mice but also in treatment-resistant depression model mice. However, because i.c.v. administration is very invasive, research is progressing on brain delivery using intranasal administration as a non-invasive method. After intranasal administration of the drug, there are two routes to the brain. That of direct delivery from the paracellular route of olfactory epithelium to the brain via the olfactory bulb has been studied, and that of systemic absorption via the paracellular route of respiratory epithelium has been put to practical use. The high degree of vascularization and permeability of the nasal mucosa enables drug delivery via the paracellular route that leads to systemic delivery. Therefore, suppressing systemic absorption may increase drug delivery to brain, so we focused on the transcellular route. We created a GLP-2 derivative by adding cell-penetrating peptides (CPP) and penetration accelerating sequences (PAS), which are reported to provide efficient intracellular uptake, to GLP-2. However, to deliver GLP-2 by the transcellular route, GLP-2 must not only be taken up into cells but also move out of the cells. We investigated in vitro and in vivo function of PAS-CPP-GLP-2 to enable the translocation of GLP-2 directly from the nose to the brain. Derivatization of PAS-CPP-GLP-2 prevented its degradation. In the evaluation of intracellular dynamics, PAS-CPP-GLP-2 enhanced cellular uptake by macropinocytosis with CPP and promoted escape from endosomal vesicles by PAS. This study also showed that PAS-CPP-GLP-2 can move out of cells. Furthermore, only this PAS-CPP-GLP-2 showed an antidepression-like effect within 20 min of intranasal administration. Intranasal administered PAS-CPP-GLP-2 surprisingly showed the effect at the same dose with i.c.v. administration, but intravenous administered PAS-CPP-GLP-2 did not show the effect. These results suggested that PAS-CPP-GLP-2 can be efficiently delivered from the nose to the CNS and show a pharmacological effect, demonstrating the usefulness of PAS and CPP for nose-to-brain delivery of GLP-2.

GLP-2 restores impairments in spatial working memory and hippocampal LTD via the MEK/ERK pathway in juvenile-onset diabetes rats.

Type 1 diabetic animal models, generated by injecting streptozotocin (STZ), have been widely used in research. We previously reported that juvenile-onset diabetes mellitus (JDM) rats, which were prepared by administering STZ to 17-day-old rats, developed cognitive impairments and hippocampal synaptic plasticity deficiencies, which were restored by glucagon-like peptide-1 (GLP-1). GLP-1 and GLP-2 are simultaneously derived from proglucagon and act through their own specific receptors. The present study was performed to investigate the potential of GLP-2 in JDM rats. The results obtained demonstrated that GLP-2 restored impairments in spatial working memory and hippocampal long-term depression (LTD) in JDM rats, and that the MEK1/2 inhibitor, U0126, inhibited this recovery. Therefore, GLP-2 has potential in the treatment of cognitive deficits in childhood-onset diabetes.

Cognitive and hippocampal synaptic profiles in monosodium glutamate-induced obese mice.

Obesity is a growing worldwide public health issue and is associated with a range of comorbidities, including cognitive deficits. The present study investigated synaptic changes in the hippocampus during the development of obesity. The treatment of newborn mice with monosodium-L-glutamate (MSG, 2 mg/g) induced obesity and recognition memory deficits in the novel object recognition (NOR) test at 16-17 weeks, but not at 8-9 weeks. Hippocampal synaptic plasticity, including long-term potentiation (LTP) and long-term depression (LTD), and excitatory synaptic transmission at Schaffer collateral-CA1 (SC-CA1) synapses were compared between MSG-treated mice and age-matched control mice. LTP and fiber volley amplitudes were enhanced in MSG-treated mice at 16-17 weeks, but not at 8-9 weeks. Furthermore, the strength of paired-pulse facilitation (PPF) changed in MSG-treated mice at 16-17 weeks, but not at 8-9 weeks. These results suggest that enhanced LTP in the SC-CA1 synapses of MSG-induced obese mice involves presynaptic rather than postsynaptic mechanisms.

Oxytocin reverses Aß-induced impairment of hippocampal synaptic plasticity in mice.

AIM: Oxytocin, a peptide hormone synthesized in the hypothalamic paraventricular nucleus, has been reported to participate in the regulation of learning and memory performance. However, no report has demonstrated the effect of oxytocin on the amyloid-beta (Aß)-induced impairment of synaptic plasticity. In this study, we examined the effects of oxytocin on the Aß-induced impairment of synaptic plasticity in mice. METHODS: To investigate the effect of oxytocin on synaptic plasticity, we prepared acute hippocampal slices for extracellular recording and assessed long-term potentiation (LTP) with perfusion of the Aß active fragment (Aß25-35) in the absence and presence of oxytocin. RESULTS: We found that oxytocin reversed the impairment of LTP induced by Aß25-35 perfusion in the mouse hippocampus. These effects were blocked by pretreatment with the selective oxytocin receptor antagonist L-368,899. Furthermore, the treatment with the ERK inhibitor U0126 and selective Ca2+-permeable AMPA receptor antagonist NASPM completely antagonized the effects of oxytocin. CONCLUSION: This is the first report to demonstrate that oxytocin could reverse the effects of Aß on hippocampal LTP in mice. We propose that ERK phosphorylation and Ca2+-permeable AMPA receptors are involved in this effect of oxytocin.

Glucagon-like peptide-2 rescues memory impairments and neuropathological changes in a mouse model of dementia induced by the intracerebroventricular administration of streptozotocin.

Glucagon-like peptide 2 (GLP-2) is derived from the proglucagon gene expressed in the intestines, pancreas and brain. Our previous study showed that GLP-2 improved lipopolysaccharide-induced memory impairments. The current study was designed to further investigated the potential of GLP-2 in memory impairment induced by intracerebroventricular administration of streptozotocin (ICV-STZ) in mice, which have been used as an animal model of sporadic Alzheimer's disease (AD). STZ was administered on alternate days (Day-1 and Day-3) in order to induce dementia in male ddY mice. ICV-STZ-treated mice were administered GLP-2 (0.6 µg/mouse, ICV) for 5 days from 14 days after the first ICV administration of STZ. In these mice, we examined spatial working memory, the biochemical parameters of oxidative stress, or neurogenesis. The GLP-2 treatment restored spatial working memory in ICV-STZ-treated mice. ICV-STZ-treated mice showed markedly increased thiobarbituric acid reactive species (TBARS) and decreased glutathione (GSH) levels, and GLP-2 significantly restored these ICV-STZ-induced changes. GLP-2 also significantly restored neurogenesis in the subgranular zone of the dentate gyrus in ICV-STZ-treated mice. We herein demonstrated that GLP-2 significantly restored ICV-STZ-induced memory impairments as well as biochemical and histopathological alterations, and accordingly, propose that the memory restorative ability of GLP-2 is due to its potential to reduce oxidative stress.

Selective agonists of the δ-opioid receptor, KNT-127 and SNC80, act differentially on extinction learning of contextual fear memory in mice.

We reported previously that KNT-127 and SNC80, selective agonists of the δ-opioid receptor (DOP), had potent anxiolytic-like effects in rodents. In this study, we evaluated whether KNT-127 and SNC80 influence extinction learning of contextual fear memory in the mice fear conditioning test. On day 1, the mice were contextually conditioned with eight trials (footshock; 0.8 mA, 1-s, 30-s interval). On day 2, the mice were re-exposed to the conditioning chamber for 6 min as an extinction training (re-exposure 1), 30 min after drug administration. On day 3, the mice were re-exposed to the chamber for 6 min as a memory testing (re-exposure 2). In re-exposure 1, KNT-127 and SNC80 significantly reduced the freezing behavior. In re-exposure 2, KNT-127, but not SNC80, significantly reduced the freezing behavior. These effects of KNT-127 were antagonized by the DOP antagonist naltrindole. KNT-127 increased the phosphorylated ERK levels in the amygdala and hippocampus, but not in the medial prefrontal cortex 60 min after re-exposure 1. These results suggest that both KNT-127 and SNC80 produced anxiolytic-like effects in the re-exposure 1, however, in contrast to SNC80, KNT-127 facilitated extinction learning of contextual fear memory in the re-exposure 2. Further, we suggest that amygdaloid and hippocampal MAPK/ERK signaling serves as the key mediators of the enhancement of extinction learning of contextual fear memory via DOPs after KNT-127 treatment. We propose that, although the DOP agonists KNT-127 and SNC80 produce anxiolytic-like effects on contextually conditioned fear, these drugs have different mechanisms on extinction learning of contextual fear memory.

[Improving Effects of Peptides on Brain Malfunction and Intranasal Delivery of Those Derivatives to the Brain].

This review focuses on the anti-dementia and antidepressant-like effects of peptides including glucagon-like peptide (GLP)-1, GLP-2, neuromedin U (NmU), and oxytocin, and the intranasal delivery of these peptides to the brain. Intracerebroventricularly administered GLP-1, NmU, and oxytocin improved impairment of learning and memory in mice treated with lipopolysaccharide or ß-amyloid protein. GLP-1 also improved impairment of learning and memory in juvenile diabetes model rats. On the other hand, GLP-2 exhibited antidepressant-like effects in mice during the forced-swim test, which were associated with 5-HT1A, α2, ß1, and D2 receptors. GLP-2 also exerted antidepressant-like effects in adrenocorticotropic hormone (ACTH)-treated mice through restoration of the hypothalamic-pituitary-adrenal-axis and neurogenesis in the subgranular zone of the dentate gyrus. Because intracerebroventricular administration is invasive and the peptides are unable to penetrate the blood-brain barrier, we introduced our new method of intranasal administration to deliver the peptides to the brain. We prepared a GLP-2 derivative containing cell-penetrating peptides (CPPs) and a penetration accelerating sequence (PAS). Intranasally administered PAS-CPPs-GLP-2 was distributed throughout the brain, and exhibited antidepressant-like effects in both naive and ACTH-treated mice. The derivatives of GLP-1, NmU, and oxytocin with the PAS and CPPs were also distributed throughout the brain after intranasal administration, and improved impairment of learning and memory. We confirmed that our peptide derivatives were effectively delivered into the brain by intranasal administration. As such, these derivatives may be useful for the clinical treatment of psychiatric and neurological diseases.

Pharmacological evidence for the relationship between the NMDA receptor and nitric oxide pathway and the antidepressant-like effects of glucagon-like peptide-2 in the mouse forced-swim test.

We previously demonstrated that glucagon-like peptide-2 (GLP-2) exerted antidepressant-like effects in mice. The aim of the present study was to investigate the relationship between N-methyl-D-aspartate (NMDA) receptor-nitric oxide-cyclic guanosine monophosphate (NO-cGMP) pathway and the antidepressant-like effects of GLP-2 in the forced-swim test (FST) in mice. Intracerebroventricularly administered GLP-2 (3 µg/mouse) decreased the immobility time in the FST. The pretreatment of mice with l-arginine (750 mg/kg, i.p.), a substrate for nitric oxide synthase, sildenafil (5 mg/kg, i.p.), a phosphodiesterase 5 inhibitor, or d-serine (300 mg/kg, i.p.), a NMDA receptor co-agonist, inhibited the antidepressant-like effects of GLP-2 (3 µg/mouse) in the FST. Meanwhile, l-nitroarginine methyl ester (10 mg/kg, i.p.), a non-specific nitric oxide synthase (NOS) inhibitor, 7-nitroindazole (30 mg/kg, i.p.), a neuronal NOS inhibitor, methylene blue (10 mg/kg, i.p.), an inhibitor of both NOS and soluble guanylate cyclase (sGC), ODQ (30 pmol/site, i.c.v.), a sGC inhibitor, or MK-801 (0.05 mg/kg, i.p.), an NMDA receptor antagonist, in combination with a sub-effective dose of GLP-2 (1.5 µg/mouse) also decreased the immobility time in the FST. The present study provided evidence for the synergistic antidepressant-like effects of GLP-2 and inhibition of the NMDA receptor-l-arginine-NO-cGMP pathway in the FST, thereby contributing to our understanding of the mechanisms underlying the antidepressant-like effects of GLP-2.

Systemic administration of a delta opioid receptor agonist, KNT-127, facilitates extinction learning of fear memory in rats.

Strategies to facilitate extinction of fear memory have attracted increasing attention for enhancing the effectiveness of exposure therapy for anxiety disorders. Previously, we demonstrated that systemic administration of a delta opioid receptor agonist, KNT-127, has clear anxiolytic-like effects in rats, without impairing memory. These observations led us to hypothesize that KNT-127 might be an appropriate therapeutic agent for anxiety disorders when combined with exposure therapy. In the present study, we demonstrate that KNT-127 (3 mg/kg) facilitates extinction learning of fear memory using the contextual fear conditioning test. As expected, a partial agonist at the glycine-binding site on the glutamatergic N-methyl-d-aspartate receptor, d-cycloserine (15 mg/kg), facilitated extinction learning of contextual fear in rats. In contrast, a benzodiazepine anxiolytic, diazepam (1 mg/kg), impaired the fear extinction learning. Interestingly, the facilitatory effect of KNT-127 on extinction learning was observed not only after a 10-min re-exposure, but also after a much shorter (2-min) re-exposure to the context, while d-cycloserine was ineffective at facilitating extinction when a short-duration exposure was given. Our findings may suggest that administration of a delta opioid receptor agonist might have therapeutic efficacy when combined with exposure therapy for treating a range of anxiety disorders.

A delta opioid receptor agonist, KNT-127, in the prelimbic medial prefrontal cortex attenuates glial glutamate transporter blocker-induced anxiety-like behavior in mice.

We previously reported that systemic administration of a delta opioid receptor (DOP) agonist, KNT-127, produced a potent anxiolytic-like effect in rats. Interestingly, DOPs are highly distributed in the prelimbic medial prefrontal cortex (PL-PFC). In the present study, we investigated the effect of KNT-127 co-perfusion in the PL-PFC on anxiety-like behavior in mice, induced by a glial glutamate transporter inhibitor, (3S)-3-[[3-[[4-(Trifluoromethyl)benzoyl]amino]phenyl]methoxy]-l-aspartic acid (TFB-TBOA). Extracellular glutamate levels were measured in male C57BL/6N mice by in vivo microdialysis high-performance liquid chromatography/electrochemical detection, with behavior simultaneously assessed in the open field test. As expected, extracellular glutamate levels were significantly increased, and anxiety-like behavior was induced after local perfusion of TFB-TBOA in the PL-PFC. Uniquely, co-perfusion of KNT-127 in the PL-PFC diminished anxiety-like behavior induced by TFB-TBOA without affecting extracellular glutamate levels. Further, the effect of KNT-127 on anxiety-like behavior was antagonized by a selective DOP antagonist, naltrindole, suggesting that KNT-127 acts via DOPs. These findings do not support our preconceived hypothesis that KNT-127 in PL-PFC produces an anxiolytic-like effect via suppression of glutamatergic transmission. Hence, further studies are necessary to understand the mechanisms of DOP agonist-induced anxiolytic-like effects in the PL-PFC.

Administration of a delta opioid receptor agonist KNT-127 to the basolateral amygdala has robust anxiolytic-like effects in rats.

RATIONALE: We previously reported that systemic administration of a selective delta opioid receptor (DOP) agonist, KNT-127, produced potent anxiolytic-like effects in rats. Interestingly, DOPs are highly distributed in the basolateral region of the amygdala (BLA). OBJECTIVES: In this study, we investigated the effect of intra-BLA administration of KNT-127 on anxiety-like behaviors in rats. METHODS AND RESULTS: In the elevated plus maze test, bilateral injection of KNT-127 into the BLA significantly and dose-dependently increased time spent in the open arms. The magnitude of KNT-127 (0.08 µg/0.2 µl)-induced anxiolytic-like effects was similar to muscimol (0.1 µg/0.2 µl), which is a selective agonist for the gamma amino butyric acid type A receptors. Further, anxiolytic-like effects of KNT-127 were abolished by pretreatment with naltrindole, a selective DOP antagonist, suggesting that KNT-127-induced anxiolytic-like effects are mediated by DOPs. These anxiolytic-like effects were confirmed using another innate anxiety model, the open field test. Interestingly, intra-BLA administration of KNT-127 also induced anxiolytic-like effects in the contextual fear conditioning test. Moreover, these effects were also abolished by naltrindole pretreatment. Finally, we demonstrated that intra-BLA administration of KNT-127 facilitates extinction learning of contextual fear in conditioned rats. CONCLUSIONS: Altogether, our findings clearly demonstrate that intra-BLA administration of KNT-127 in rats has robust anxiolytic-like effects not only in innate anxiety-like behavioral tests but also in the contextual fear conditioning test.

Hydroxyobtustyrene protects neuronal cells from chemical hypoxia-induced cell death.

Hydroxyobtustyrene is a derivative of cinnamyl phenol isolated from Dalbergia odorifera T. Chen. The heartwood, known as 'JiangXiang', is a traditional Chinese medicine. Previous studies showed that hydroxyobtustyrene inhibited the biosynthesis of prostaglandins, which are mediators of neuronal cell death in ischemia. However, it currently remains unclear whether hydroxyobtustyrene protects neurons against ischemic stress. In the present study, we investigated the protective effects of hydroxyobtustyrene against sodium cyanide (NaCN)-induced chemical ischemia. Hippocampal neurons were cultured from the cerebral cortices of E18 Wistar rats. The effects of hydroxyobtustyrene on neuronal survival and trophic effects were estimated under lower and higher cell density conditions. After the treatment of 1 mM NaCN with or without hydroxyobtustyrene, an MTT assay, Hoechst staining, and immunocytochemistry for cyclooxygenase (COX)-2 were performed. Hydroxyobtustyrene increased cell viability under lower, but not normal density conditions. Neither the neurite number nor the length was influenced by hydroxyobtustyrene. NaCN significantly decreased viability and increased fragmentation in cell nuclei, and these changes were prevented by hydroxyobtustyrene. Moreover, NaCN increased the number of COX-2-positive neurons, and this was significantly prevented by the co-treatment with hydroxyobtustyrene. Therefore, hydroxyobtustyrene protected cultured hippocampal neurons against NaCN-induced chemical ischemia, which may be mediated by the inhibition of COX-2 production.

Effects of centrally administered glucagon-like peptide-2 on blood pressure and barosensitive neurons in spontaneously hypertensive rats.

The central administration of glucagon-like peptide-2 (GLP-2) decreases blood pressure in rats. In the present study, we investigated the hypotensive effects of GLP-2 using spontaneously hypertensive rats (SHRs), an animal model of hypertension. The central administration of GLP-2 (0.6 µg) decreased mean arterial pressure (MAP) in SHRs (-24.1 ±â€¯4.5%; P < 0.05), but not in normotensive Wistar-Kyoto (WKY) rats (-10.6 ±â€¯7.4%; P > 0.05), whereas GLP-2 (6 µg) decreased MAP in WKY rats (-23.5 ±â€¯4.2%; P < 0.05) and SHRs (-46.7 ±â€¯11.6%; P < 0.01) under anesthesia with urethane and α-chloralose. Histological analyses revealed that the central administration of GLP-2 (6 µg) induced Fos immunoreactivity (Fos-IR) in the hypothalamic and medullary areas in WKY rats and SHRs. However, the distribution of Fos-IR in GABAergic neurons in the rostral ventrolateral medulla (RVLM) differed between WKY rats and SHRs. GLP-2 directly modulated the excitability of RVLM neurons in brainstem slices from SHRs, but not WKY rats. These results suggest that neuronal activity through the activation of GLP-2 receptors in the RVLM contributes to lowering blood pressure in SHRs.

Endogenous acetylcholine regulates neuronal and astrocytic vascular endothelial growth factor expression levels via different acetylcholine receptor mechanisms.

Vascular endothelial growth factor (VEGF), a signaling molecule involved in angiogenesis, plays an important role in neuroprotection and neurogenesis. In the present study, we aimed to elucidate the mechanisms underlying endogenous acetylcholine (ACh)-induced VEGF expression in neurons and astrocytes, and identify the neuronal cells contributing to its expression in the medial septal area, a nuclear origin of cholinergic neurons mainly projecting to the hippocampus. The mRNA expression and secretion of VEGF were measured by RT-PCR and ELISA using mouse primary cultured cortical neurons and astrocytes. VEGF expression in the medial septal area was assessed by RT-PCR and immunostaining using mice treated with tacrine [9-amino-1,2,3,4-tetrahydro-acridine HCl (THA); 2.5 mg/kg, i.p.] once daily for 7 days. The THA treatment increased VEGF mRNA expression in neurons in a manner that was reversed by mecamylamine, a nicotinic ACh receptor (AChR) antagonist, whereas in mouse primary cultured astrocytes, carbachol, but not THA dose-dependently increased VEGF mRNA expression and secretion in a manner that was inhibited by scopolamine, a muscarinic AChR inhibitor. In in vivo studies, the administration of THA significantly increased the expression of VEGF in medial septal cholinergic neurons and the effects of THA were significantly blocked by mecamylamine. THA also significantly increased the expression levels of a phosphorylated form of VEGF receptor 2 (p-VEGFR2), an activated form of VEGFR2. The present results suggest that endogenous ACh plays an up-regulatory role for VEGF expression in neurons and astrocytes via different mechanisms. Moreover, endogenous ACh-induced increases in VEGF levels appear to activate VEGFR2 on medial septal cholinergic neurons via an autocrine mechanism.

The delta opioid receptor agonist KNT-127 in the prelimbic medial prefrontal cortex attenuates veratrine-induced anxiety-like behaviors in mice.

We previously reported that systemic administration of the selective delta opioid receptor (DOP) agonist KNT-127 produces potent anxiolytic-like effects in rats. Although a higher distribution pattern of DOPs was reported in the prelimbic medial prefrontal cortex (PL-PFC) of rodents, the role of DOPs in PL-PFC and in anxiolytic-like effects have not been well examined. Recently, we demonstrated that activation of PL-PFC with the sodium channel activator veratrine increases glutamatergic neurotransmission and produces anxiety-like behaviors in mice. Therefore, we investigated the effects of co-perfusion with KNT-127 in PL-PFC on veratrine-induced anxiety-like behaviors in mice. We also simultaneously measured extracellular glutamate and GABA levels. In addition, we assessed the effect of KNT-127 on the expression of c-Fos in sub-regions of the amygdala. Extracellular glutamate levels were measured in seven-week-old male C57BL/6N mice using an in vivo microdialysis-HPLC/ECD system, and behaviors were assessed simultaneously in an open field test. Basal levels of glutamate were measured by collecting samples every 10min for 60min. The drug-containing medium was perfused for 30min, and the open field test was performed during the last 10min of drug perfusion. After drug treatments, the perfusion was switched from drug-containing medium to control medium without drugs and samples were collected for another 90min. KNT-127 co-perfusion completely diminished veratrine-induced anxiety-like behaviors and attenuated the veratrine-induced increase in extracellular glutamate levels in PL-PFC. Interestingly, KNT-127 perfusion alone in PL-PFC did not affect anxiety-like behaviors. Local perfusion of veratrine in PL-PFC induced c-Fos immunoreactivity in sub-regions of amygdala. Co-perfusion of KNT-127 diminished c-Fos expression. Here we demonstrate that the DOP agonist KNT-127 in PL-PFC attenuates veratrine-induced anxiety-like behaviors in mice. These effects may be caused by the presynaptic suppression of activated glutamatergic transmission in PL-PFC, which projects to sub-regions of the amygdala. We propose that compounds like KNT-127, which inhibit glutamatergic transmission in PL-PFC, are candidates for novel anxiolytics.