Hiroo Ogura

Mice lacking bombesin receptor subtype-3 develop metabolic defects and obesity

Mammalian bombesin-like peptides are widely distributed in the central nervous system as well as in the gastrointestinal tract, where they modulate smooth-muscle contraction, exocrine and endocrine processes, metabolism and behaviour. They bind to G-protein-coupled receptors on the cell surface to elicit their effects. Bombesin-like peptide receptors cloned so far include, gastrin-releasing peptide receptor (GRP-R), neuromedin B receptor (NMB-R), and bombesin receptor subtype-3 (BRS-3). However, despite the molecular characterization of BRS-3, determination of its function has been difficult as a result of its low affinity for bombesin and its lack of an identified natural ligand. We have generated BRS-3-deficient mice in an attempt to determine the in vivo function of the receptor. Mice lacking functional BRS-3 developed a mild obesity, associated with hypertension and impairment of glucose metabolism. They also exhibited reduced metabolic rate, increased feeding efficiency and subsequent hyperphagia. Our data suggest that BRS-3 is required for the regulation of endocrine processes and metabolism responsible for energy balance and adiposity. BRS-3-deficient mice provide a useful new model for the investigation of human obesity and associated diseases.

Facilitation of NMDAR-Independent LTP and Spatial Learning in Mutant Mice Lacking Ryanodine Receptor Type 3

To evaluate the role in synaptic plasticity of ryanodine receptor type 3 (RyR3), which is normally enriched in hippocampal area CA1, we generated RyR3-deficient mice. Mutant mice exhibited facilitated CA1 long-term potentiation (LTP) induced by short tetanus (100 Hz, 100 ms) stimulation. Unlike LTP in wild-type mice, this LTP was not blocked bythe NMDA receptor antagonist D-AP5 but was partially dependent on L-type voltage-dependent Ca2+ channels (VDCCs) and metabotropic glutamate receptors (mGluRs). Long-term depression (LTD) was not induced in RyR3-deficient mice. RyR3-deficient mice also exhibited improved spatial learning on a Morris water maze task. These results suggest that in wild-type mice, in contrast to the excitatory role of Ca2+ influx, RyR3-mediated intracellular Ca2+ ([Ca2+]i) release from endoplasmic reticulum (ER) may inhibit hippocampal LTP and spatial learning.

Orexin-A (hypocretin-1) is possibly involved in generation of anxiety-like behavior

Orexins (hypocretins) are neuropeptides expressed specifically in neurons in the lateral hypothalamic area and are known to be involved in the regulation of vigilance and feeding behavior. However, the relationship between orexin and emotional behaviors like anxiety is still poorly understood. Therefore, in this report we evaluated the effect of intracerebroventricular injection of orexin-A in two major anxiety tests, the light-dark exploration test (mouse) and the elevated plus-maze test (mouse, rat). Orexin increased time spent in the dark compartment in the light-dark test and time spent on the closed arms in the elevated plus-maze test. These results were not caused by a hypothetical sedative or activity-inducing effect of orexin-A because spontaneous locomotor activity did not alter upon orexin-A application under novel conditions. We therefore suggest an anxiogenic effect of orexin-A. To our knowledge, this is the first report about a relationship between orexin-A and anxiety.

Chronic Intracerebroventricular Administration of Relaxin-3 Increases Body Weight in Rats

Bolus-administered intracerebroventricular (ICV) relaxin-3 has been reported to increase feeding. In this study, to examine the role of relaxin-3 signaling in energy homeostasis, we studied the effects of chronically administered ICV relaxin-3 on body weight gain and locomotor activity in rats. Two groups of animals received vehicle or relaxin-3 at 600 pmol/head/day, delivered with Alzet osmotic minipumps. In animals receiving relaxin-3, food consumption and weight gain were statistically significantly higher than those in the vehicle group during the 14-day infusion. During the light phase on days 2 and 7 and the dark phase on days 3 and 8, there was no difference in locomotor activity between the two groups. Plasma concentrations of leptin and insulin in rats chronically injected with relaxin-3 were significantly higher than in the vehicle-injected controls. These results indicate that relaxin-3 up-regulates food intake, leading to an increase of body weight and that relaxin-3 antagonists might be candidate antiobesity agents.

Impairment of hippocampal long-term depression and defective spatial learning and memory in p35-/- mice

Cdk5 (cyclin‐dependent kinase 5) activity is dependent upon association with one of two neuron‐specific activators, p35 or p39. Genetic deletion of Cdk5 causes perinatal lethality with severe defects in corticogenesis and neuronal positioning. p35–/– mice are viable with milder histological abnormalities. Although substantial evidence implicates Cdk5 in synaptic plasticity, its role in learning and memory has not been evaluated using mutant mouse models. We report here that p35–/– mice have deficiencies in spatial learning and memory. Close examination of hippocampal circuitry revealed subtle histological defects in CA1 pyramidal cells. Furthermore, p35–/– mice exhibit impaired long‐term depression and depotentiation of long‐term potentiation in the Schaeffer collateral CA1 pathway. Moreover, the Cdk5‐dependent phosphorylation state of protein phosphatase inhibitor‐1 was increased in 4‐week‐old mice due to increased levels of p39, which co‐localized with inhibitor‐1 and Cdk5 in the cytoplasm. These results demonstrate that p35‐dependent Cdk5 activity is important to learning and synaptic plasticity. Deletion of p35 may shift the substrate specificity of Cdk5 due to compensatory expression of p39.

Apoptotic cell death of cultured cerebral cortical neurons induced by withdrawal of astroglial trophic support

Peripheral neurons which depend on NGF for their survival undergo apoptosis after NGF deprivation. However, a convenient in vitro method for assessing the programmed cell death of the central neurons has not been established, because the dependence of particular central neurons on neurotrophic factors has been clarified only for small populations of neurons. Based on the fact that cortical neurons survive in culture for many weeks in the presence of astroglial cells, we have established an in vitro cell death model in which the neurons die through apoptosis. Cortical neurons were maintained on a cover slip for 1 week on top of astroglial cells, and then cell death was induced by separation of the neurons from the astroglial cells. The cortical neurons died within 2-4 days. Nuclei of the dying neurons showed the morphological features of apoptosis, and DNA fragmentation was observed by the TUNEL method and by in situ nick translation (ISNT) staining. The cell death was significantly suppressed by neurotrophic factors, NT-3, NT-4, BDNF, and GDNF, but not by NGF. The neuronal survival was prolonged, as in the case of peripheral neurons, by bFGF, elevated potassium, cAMP, forskolin, and metabotropic glutamate receptor agonist. The cell death was inhibited by inhibitors of interleukin-1 beta-converting enzyme and CPP32. CPP32-like proteolytic activity was increased prior to the appearance of apoptotic cells. These results suggest that cortical neurons die after separation from glial cells through apoptosis caused by deprivation of neurotrophic factors produced by the astroglial cells.

Estrogen induces a rapid increase of calcium-calmodulin-dependent protein kinase II activity in the hippocampus

Molecular genetics experiments using gene targeting and transgenic technology demonstrated the importance of alpha-calcium-calmodulin-dependent protein kinase II (alphaCaMKII) in long-term potentiation (LTP) and memory. Little information is available though on how CaMKII activity may be regulated in vivo. We show that estradiol benzoate activates CaMKII in a dose and time-dependent manner in mouse hippocampus after 30 min stimulation. The effect of estrogen is via a very rapid nongenomic mechanism that is blocked in vitro in hippocampal primary neurons by the pure estrogen receptor antagonist, ICI 182,780. These results suggest that estrogen action in the hippocampus is linked to CaMKII activation.

Comparison of mice deficient in the high- or low-affinity neurotensin receptors, Ntsr1 or Ntsr2, reveals a novel function for Ntsr2 in thermal nociception.

Neurotensin (NT) is a neuropeptide that induces a wide range of biological activities including hypothermia and analgesia. Such effects are mediated by the NT receptors Ntsr1, Ntsr2 and Ntsr3, although the involvement of each receptor in specific NT functions remains unknown. To address nociceptive function in vivo, we generated both Ntsr1-deficient and Ntsr2-deficient mice. In addition, histochemical analyses of both Ntsr1 and Ntsr2 mRNAs were performed in the mouse brain regions involved in NT-related nociception. The expression of Ntsr2 mRNA was greater than that of Ntsr1 in the periaqueductal gray (PAG) and the rostral ventral medulla (RVM). The mutant and control mice were subjected to the examination of thermal nociception, and in the hot plate test, a significant alteration in jump latency was observed in Ntsr2-deficient mice compared to Ntsr1-deficient or wild-type control mice. Latencies of tail flick and hind paw licking of the mutant mice were not affected compared to control mice. These results suggest that Ntsr2 has an important role in thermal nociception compared to Ntsr1, and that these mutant mice may represent a useful tool for the development of analgesic drugs.

Behavioral abnormalities of Zic1 and Zic2 mutant mice: implications as models for human neurological disorders.

Zic1 and Zic2 encode closely related zinc finger proteins expressed in dorsal neural tube and its derivatives. In previous studies, we showed that the homozygous Zic1 null mutation (Zic1−/−) results in cerebellar malformation with severe ataxia and that holoprosencephaly and spina bifida occur in homozygotes for Zic2 knockdown mutation (Zic2kd/kd). Since human ZIC2 haploinsufficiency is a cause of holoprosencephaly, the Zic2kd/kd mice are regarded as an animal model for holoprosencephaly in humans. In this study, the behavioral characteristics of the Zic1 and Zic2 mutant mice were investigated in heterozygotes (Zic1−/+ or Zic2kd/+), and significant abnormalities were found in the hanging, spontaneous locomotor activity, stationary rod (Zic1−/+), acoustic startle response, and prepulse inhibition tests (Zic2kd/+). The abnormalities in the Zic1−/+ mice may be explained in part by the hypotonia caused by hypoplasia of the cerebellar anterior vermis, and these mice are regarded as a model of Joubert syndrome. In contrast, the sensorimotor gating abnormality in the Zic2kd/+ mice may be attributable to the presumed abnormality in the dorsomedial forebrain, which was strongly affected in the Zic2kd/kd mice. Zic2kd/+ mice can serve as a model for diseases involving sensorimotor gating abnormalities, such as schizophrenia.

Motor discoordination in mutant mice heterozygous for the type 1 inositol 1,4,5-trisphosphate receptor.

In the brain, the type 1 inositol 1,4,5-trisphosphate receptor (IP3R1) is a major subtype of receptors for inositol 1,4,5-trisphosphate, which mediates the release of calcium from intracellular stores. The motor function of knockout mice heterozygous for IP3R1 (IP3R1+/-) was assessed. An impairment of motor coordination was observed in IP3R1+/- mice in the rotating rod test. There was no observable difference between genotypes in spontaneous motor activity, grip strength, the hanging test, or walking pattern. These results suggest that IP3R1 plays a substantial role in motor coordination.