Neurobehavioral Differences Between Mice Receiving Distinct Neuregulin Variants as Neonates; Impact on Sensitivity to MK-801.

Neuregulin-1 (NRG1) is a well-recognized risk gene for schizophrenia and is often implicated in the neurodevelopmental hypothesis of this illness. Alternative splicing and proteolytic processing of the NRG1 gene produce more than 30 structural variants; however, the neuropathological roles of individual variants remain to be characterized. On the basis of the neurodevelopmental hypothesis of schizophrenia, we administered eNRG1 (0.1~1.0 µg/g), a core epidermal growth factor-like (EGF) domain common for all splicing NRG1 variants, to neonatal mice and compared their behavioral performance with mice challenged with a full mature form of type 1 NRG1 variant. During the neonatal stage, recombinant eNRG1 protein administrated from the periphery passed the blood-brain barrier and activated its receptor (ErbB4) in the brain. In adults, the mice receiving the highest dose exhibited lower locomotor activity and deficits in prepulse inhibition and tonedependent fear learning, although the hearing reduction of the eNRG1-treated mice may explain these behavioral deficits. Neonatal eNRG1 treatment also significantly potentiated MK-801-driven locomotor activity in an eNRG1 dose-dependent manner. In parallel eNRG1 treatment enhanced MK-801-driven c-Fos induction and decreased immunoreactivity for NMDA receptor subunits in adult brain. In contrast, mice that had been treated with the same molar dose of a full mature form of type 1 NRG1 as neonates did not exhibit hypersensitivity to MK-801. However, both animal models exhibited similar hypersensitivity to methamphetamine. Collectively, our findings suggest that aberrant peripheral NRG1 signals during neurodevelopment alter later behavioral traits and auditory functions in the NRG1 subtype-dependent manner.

Identification of differentially expressed genes in rat hippocampus after transient global cerebral ischemia using subtractive cDNA cloning based on polymerase chain reaction.

BACKGROUND AND PURPOSE: The purpose of this study is to identify new molecules that play important roles in the phenomena that occur in the hippocampus after transient global cerebral ischemia, as clues to better understanding of the mechanisms. METHODS: A subtractive cDNA library was established by suppression subtractive hybridization of rat hippocampal tissues after transient global cerebral ischemia. With differential screening of the library, upregulated fragments were identified. The mRNA expression levels of selected genes were measured with semiquantitative reverse transcriptase polymerase chain reaction (PCR). RESULTS: Among more than 100 isolated fragments, approximately half were determined to be identical to known sequences. The rest showed high homology to known sequences, and only 2 did not exhibit homology to any known sequences. The expression of 5 genes identified in this study increased in 24 hours after ischemia to a level twice as high as that in sham-operated controls. These included furin, prosaposin, synaptotagmin IV, heat shock protein 105, and the neutral and basic amino acid transporter (NBAT). The increases in the mRNA expression levels of the genes except NBAT, as revealed by semiquantitative reverse transcription PCR, were statistically significant at both 6 and 24 hours after ischemia. CONCLUSIONS: Genes isolated are thought to be associated with production of proteins necessary for degeneration, neuroprotection, and reconstruction of neurons. How the expression of these genes relates to functional changes after ischemia remains to be determined. PCR-based subtractive cDNA cloning is demonstrated to be a useful tool for analyzing in vivo gene expression in animal ischemia models.

The optimal stimulation site for chronic stimulation of the subthalamic nucleus in Parkinson's disease.

To determine the optimal stimulation site within the subthalamic nucleus (STN), monopolar stimulation of four electrode contacts and the resulting effects on parkinsonian symptoms were evaluated in 10 consecutive patients. The UPDRS score for rigidity and akinesia improved significantly after stimulation at each of the contacts, compared to the pre-evaluation state (Fisher's test, p < 0.05). The most significant improvement was obtained after stimulation at contact-2 (rigidity: 74.4 +/- 20.4%, akinesia: 53.7 +/- 14.3%) (Fisher's test, p < 0.001). Contact-2 was located at the dorsal border of the STN at a mean distance of 0.3 +/- 0.7 mm. DBS at the dorsal border of the STN, where the stimulation affects the neurons as well as their axonal fibers, produces the greatest clinical improvement in parkinsonian symptoms.

[Neurosurgical treatment for dopamine-induced dyskinesias in Parkinson's disease patients].

The effects of current neurosurgical interventions for levodopa-induced dyskiensias (DID) in Parkinson's disease are reviewed. Thalamotomy has been reported to be effective for DID when the lesions include Vo or CM-Pf nucleus, while thalamic deep brain stimulation(DBS) is less effective than thalamotomy. Both pallidotomy and pallidal DBS are probably the most effective neurosurgical treatment for DID, because they significantly improve all of the DID, including off-period dystonia, without reduction of levodopa dosage. Subthalamic DBS has no direct therapeutic effects on DID, but substantially can improve DID as a result of decreased levodopa dosage. The effects of cerebral transplantation on DID remain undefined. More researches are expected to clarify the pathophysiology of DID.

Thyrotropin regulates c-Jun N-terminal kinase (JNK) activity through two distinct signal pathways in human thyroid cells.

c-Jun N-terminal kinases (JNK) participate in cellular responses to mitogenic stimuli and environmental stresses. We investigated whether and how TSH, which promotes the proliferation and differentiation of thyroid cells, regulates JNK activity in primary cultured human thyroid cells. TSH stimulated JNK activity in cytosolic fractions of thyroid cells measured by in vitro kinase assay. A low concentration of TSH (10(-11) M) stimulated JNK activity but at a higher dose (10(-8)-10(-7) M), TSH suppressed JNK activity without any change of JNK protein level. Activation of JNK by TSH was also observed in CHO cells stably transfected with TSH receptor complementary DNA (cDNA), suggesting a ligand-receptor specific interaction. TSH stimulated JNK activity through a pertussis toxin-sensitive pathway. We next elucidated the signal transduction pathways in TSH-induced JNK activation by examining the involvement of four distinct intracellular signal molecules; protein kinase C (PKC), cAMP, Ca2+, and PI3-kinase. The stimulation of JNK by TSH was blocked by two PKC inhibitors and suppressed by 8-bromo-cAMP or forskolin. These findings demonstrate that TSH regulates JNK activity biphasically in human thyroid cells through an interaction between Gi-PKC and cAMP-PKA pathways.

Human cerebral acetylcholinesterase activity measured with positron emission tomography: procedure, normal values and effect of age.

The regional cerebral metabolic rate of [11C]N-methyl-4-piperidyl acetate, which is nearly proportional to regional cerebral acetylcholinesterase (AChE) activity, was measured by dynamic positron emission tomography in 20 healthy subjects with a wide age range (24-89 years). Quantitative measurement was achieved using a kinetic model which consisted of arterial plasma and cerebral tissue compartments. The plasma input function was obtained using thin-layer chromatography and an imaging phosphor plate system at frequent sampling intervals to catch the rapid metabolism of the tracer in the blood. The distribution of the rate constant k3, an index of AChE activity, agreed well with reported post-mortem AChE distribution in the cerebral cortex (0.067-0.097 min-1) and thalamus (0.268 min-1), where AChE activity was low to moderate. The k3 values in the striatum and cerebellum, where AChE activity was very high, did not respond linearly to AChE activity because of increased flow dependency. No significant effect of age was found on AChE activity of the cerebral cortex, suggesting that the ascending central cholinergic system is preserved in normal aging. This study has shown that quantitative measurement of enzyme activity in the living brain is possible through appropriate modelling of tracer kinetics and accurate measurement of the input function. The method should be applicable to patients with Alzheimer's disease and those with other kinds of dementia whose central cholinergic system has been reported to be disturbed.

[Assumption of the area supplying Okayama Prefecture with Cryptomeria japonica and Cupressaceae airborne pollen, and of their scattering routes].

It is very important to predict and disseminate information about the total pollen counts of both Cryptomeria japonica and Cupressaceae for patients with pollinosis. In Okayama Prefecture, we have reported that the pollen counts of both Cryptomeria japonica and Cupressaceae are influenced by the meteorological conditions in the previous July. We predicted the area supplying Okayama Prefecture with Cryptomeria japonica and Cupressaceae pollen, and also the route of airborne pollen from the meteorological conditions and a topographical map of Okayama and four neighboring prefectures. It was found that Cryptomeria japonica and Cupressaceae pollen counts at the four observation sites correlated very well with the meteorological conditions at Tsuyama weather station in Okayama prefecture. Therefore, we considered that the areas supplying Okayama prefecture with Cryptomeria japonica and Cupressaceae pollen were the central northern areas including Tsuyama, 85.7% of whose plantation areas contained Cryptomeria japonica and Chamaecyparis obtusa, and that their pollen was carried along the routes of three major rivers, R. Takahashi, R. Asahi and R. Yoshii.

Expression profile of receptor-type protein tyrosine kinase genes in the human thyroid.

Protein tyrosine kinases (PTKs) play a role in regulating the growth and differentiated functions of thyroid cells and are probably involved in tumorigenesis of papillary-type thyroid carcinoma. To better understand the roles of PTKs in the physiology and pathophysiology of the thyroid, we analyzed the expression profile of receptor-type PTKs in normal human thyroid tissues. Highly conserved regions in the catalytic domains of receptor-type PTKs were amplified by RT-PCR using degenerate oligonucleotide primers. Nucleotide sequencing of about 100 clones identified 21 PTKs, including 16 receptor type and 5 nonreceptor type; no novel PTK was identified. Insulin-like growth factor I receptor, platelet-derived growth factor receptor (PDGFR), TrkE, Axl, epidermal growth factor receptor, etc., appear to be the most abundant receptor-type PTKs in the thyroid; many of which (PDGFR, TrkE, Axl, etc.) have never previously been demonstrated to be expressed in the thyroid. The expression of messenger RNAs (mRNAs) for PDGFR, axl, and trkE in normal thyroid cells was confirmed by Northern blot analysis, and interestingly, the expression levels of PDGFR and trkE mRNAs were decreased in all three thyroid carcinoma cell lines examined (FRO, WRO, and NPA), whereas axl mRNA and protein were overexpressed in 2 of 3 thyroid carcinoma cell lines (FRO and WRO) compared with that in normal tissue. The axl gene was, however, neither amplified nor rearranged. The biological activity of the ligand for Axl, the product of growth arrest-specific gene 6 (Gas6), was then evaluated, demonstrating modest mitogenic activity in thyroid carcinoma cells overexpressing Axl. Furthermore, gas6 mRNA was expressed in FRO cells. Thus, we here identify a variety of PTKs expressed in the thyroid gland, many of which may participate in the regulation of thyroid cell function. Variable expression levels of some PTKs in normal and cancerous cells suggest that there may be an imbalance and disarray of phosphorylation events in thyroid carcinoma cells. Furthermore, Gas6 is identified as a novel growth factor for thyroid carcinoma cells overexpressing Axl receptor tyrosine kinase.

Time courses of changes in cerebral blood flow and blood-brain barrier integrity by focal proton radiation in the rat.

In order to know the pathophysiological mechanisms underlying radiation brain injury, cerebral blood flow and blood-brain barrier integrity were studied using N-isopropyl-p-[123l]iodoamphetamine (IMP) and [14C]-alpha-aminoisobutyric acid (AIB), respectively, in the rat focal proton radiation model (a single dose of 30 or 60 Gy radiation with 70 MeV proton beams). One, 2, 4, and 5.5 months after irradiation, [123l]IMP and [14C]AIB were intravenously injected and uptake of IMP and AIB in the cerebral cortex, striatum, hippocampus, and thalamus was measured. Significant decreases in IMP uptake were observed in the cerebral cortex and thalamus of the irradiated side at 4 and 5.5 months after 60 Gy irradiation; the effects at 5.5 months were more prominent than those at 4 months. AIB uptake markedly increased in all the brain regions of the irradiated side at 5.5 months after 60 Gy irradiation, and at 4 months, only in the hippocampus. The results suggest that there are dose- and time-dependent responses in radiation effects and regional differences in tissue vulnerability to radiation. Proton focal radiation model appears to be a useful model for studies of radiation brain injury in small animals such as rats.

In vivo measurement of acetylcholinesterase activity in the brain with a radioactive acetylcholine analog.

A novel method for visualization of brain acetylcholinesterase (AChE) in vivo has been developed. Following intravenous administration of a radiolabelled acetylcholine analog, N-methyl-3-piperidyl acetate, there was very good agreement between the distribution of radioactivity and AChE activity in the brain of rat and monkey. The method would be applicable for in vivo studies of human brain AChE activity in disorders of central cholinergic systems such as Alzheimer's disease.

Overexpression of the intact thyrotropin receptor in a human thyroid carcinoma cell line.

Although thyrotropin is known to regulate thyroid cell differentiation and proliferation, human thyroid carcinoma cells are relatively insensitive or resistant to TSH stimulation. The expression levels of TSH receptor are significantly lower in carcinoma tissues than in normal tissues. Furthermore, in vitro human thyroid cell growth is not regulated by TSH itself. We, therefore, isolated neomycin-resistant stable human thyroid carcinoma cell (WRO cell) transfectants overexpressing intact human TSH receptor to evaluate the functional role of TSH receptor on carcinoma cells. Southern blot analysis confirmed incorporation and amplification of human TSH receptor complementary DNA sequences into genomic DNA. Northern gel analysis and reverse transcriptase-polymerase chain reaction analysis revealed the presence of specific TSH receptor messenger RNA (4.0 kilobases), and the specific binding and the affinity of [125I]TSH on stably transfected WRO cells were demonstrated compared to wild type. Nevertheless, impaired cAMP production to transfectants by TSH was observed. cAMP production was confirmed after stimulation of both wild type and transfectants by forskolin, cholera toxin, and isoproterenol. In contrast, TSH could affect the cytoplasmic calcium mobilization immediately after the addition of TSH to WRO transfectants. These results suggest that the impairment of TSH action on human thyroid carcinoma cells is not due to a major structural abnormality of the TSH receptor, reduction in the receptor number, or receptor affinity, but much more likely due to a TSH receptor-guanyl nucleotide-binding protein coupling defect.

[Physiologically active compounds in the extracts from tochukaso and cultured mycelia of Cordyceps and Isaria].

Tochukaso is a Chinese traditional medicine composed of a fruit body of Cordyceps sinensis and its parasitic host larva. Tochukaso (C. sinensis) and the cultured mycelia of five species of Cordyceps and four species of Isaria were each extracted with hot water and examined for the inotropic effect on guinea-pig right atrium in vitro system. The extracts from C. militaris and I. felina showed a negative inotropic effect to approximately the same extent as that from Tochukaso. These three extracts also showed inhibitory action on twitch response of guinea-pig ileum and aggregation of human blood platelet. It is suggested that these activities are ascribed to the combination of adenosine, 5'-adenosine monophosphate and several other nucleic acid-related compounds, all of which have been shown to be present in the extracts.

Effects of insulin on local cerebral glucose utilization in the rat.

The effects of hyperinsulinemia on local cerebral glucose utilization were studied by the quantitative autoradiographic 2-[14C]deoxyglucose method in normal conscious rats under steady-state normoglycemic conditions. Hyperinsulinemia and a steady state of normoglycemia were achieved and maintained during the experimental period by a continuous intravenous (i.v.) infusion of insulin given simultaneously with a programmed i.v. infusion of D-glucose. Hyperinsulinemia under normoglycemic conditions did not change the average rate of glucose utilization in the brain as a whole, but significant increases in local glucose utilization were found selectively in the ventromedial, dorsomedial, and anterior hypothalamic nuclei. The results suggest that a known anatomical pathway linking the dorsomedial and anterior nuclei with the ventromedial nucleus of the hypothalamus may be physiologically activated in response to hyperinsulinemia.