Control of aversion by glycine-gated GluN1/GluN3A NMDA receptors in the adult medial habenula.

The unconventional N-methyl-d-aspartate (NMDA) receptor subunits GluN3A and GluN3B can, when associated with the other glycine-binding subunit GluN1, generate excitatory conductances purely activated by glycine. However, functional GluN1/GluN3 receptors have not been identified in native adult tissues. We discovered that GluN1/GluN3A receptors are operational in neurons of the mouse adult medial habenula (MHb), an epithalamic area controlling aversive physiological states. In the absence of glycinergic neuronal specializations in the MHb, glial cells tuned neuronal activity via GluN1/GluN3A receptors. Reducing GluN1/GluN3A receptor levels in the MHb prevented place-aversion conditioning. Our study extends the physiological and behavioral implications of glycine by demonstrating its control of negatively valued emotional associations via excitatory glycinergic NMDA receptors.

Different effects of L-, N- and T-type calcium channel blockers on striatal dopamine release measured by microdialysis in freely moving rats.

Using a microdialysis method, we have investigated effects of the voltage-dependent calcium channel blockers, verapamil, nicardipine, omega-conotoxin and flunarizine on the dopamine release and metabolism in the striatum of freely moving rat. Perfusion of verapamil (1-300 microM) and nicardipine (1-100 microM), an L-type calcium channel blocker, into the striatum through the dialysis membrane showed a dose-dependent decrease of dopamine release in the dialysate and slight increase of 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) levels. Treatment of omega-conotoxin (0.1, 1 microM), an N-type channel blocker, decreased about 50% basal dopamine release and slightly decreased DOPAC and HVA levels. Treatment with flunarizine (10 microM), an T-type channel blocker, did not affect the dopamine release and metabolism. From these data, it appears that treatments of the L- and N-type voltage-dependent calcium channel blockers in rat striatum suppress basal dopamine release, but T-type blocker does not suppress it, suggesting that L-, N- and T-type calcium channels regulate in vivo dopamine release in a different mechanism.

A functional MRI analysis of comprehension processes of Japanese sentences.

We tried to identify the mechanisms directly related to syntactic processing in an fMRI experiment using strictly controlled sets of verbal stimuli. In two conditions, center-embedding and left-branching conditions, the same sets of words were used to construct stimulus sentences, while only the word order reflecting their syntactic structure was different. The subject's task was to understand the relationship among three characters mentioned in the sentences. A difference of activation in Broca's area (BA44 and 45) between the two conditions was found. This result suggests that these areas are involved in syntax-related processing. Furthermore, it was suggested that the posterior part of the frontal lobe (BA6/9) and the inferior parietal area (BA39/40) are involved in the understanding of the relationship among the three characters mentioned in each sentence.

An inhibitor for calcineurin, FK506, blocks induction of long-term depression in rat visual cortex.

Long-term depression (LTD) of synaptic transmission, often used as an essential component in synaptic models for learning, memory and forgetting, can be produced in layer II/III of the visual cortex by a prolonged, low-frequency stimulation (LFS) of layer IV. The activation of Ca2+/calmodulin-dependent protein phosphatase, calcineurin, has been postulated to play a role in the induction of LTD. The recent introduction of a specific inhibitor for calcineurin, FK506, prompted the investigation of the involvement of this phosphatase in the induction of LTD in visual cortex. Thus, we administered FK506 at 1 microM to visual cortical slices of young rats, and found that it did not significantly affect field responses of layer II/III evoked by test stimulation of layer IV at 0.1 Hz, but prevented LTD of the responses from being induced by LFS (1 Hz for 15 min) in all the 10 slices tested. Without FK506, significant LTD was induced by the same parameters of LFS in 8 of the 12 slices. These results suggest the critical involvement of calcineurin in producing LTD in visual cortex.

Methylisobutylxanthine, a potent inhibitor of cAMP phosphodiesterase, enhances the sensitivity of Chinese hamster ovary cells to mitomycin C.

The effect of 1-methyl-3-isobutylxanthine (MIX) on the sensitivity of CHO cells to mitomycin C (MC) was examined. Treatment of cells with MIX before MC-exposure greatly enhanced the cellular sensitivity to MC, with no effect on plating efficiency. Treatment with MIX after MC-exposure had only a slight effect. The sensitivity to MC was enhanced, biphasically, with increases in the period of pretreatment with MIX. The sensitivity of cells to MC fluctuated through the cell cycle, in that they were most sensitive to MC in G1 and resistant in the G2/M phase. Flow cytometric studies revealed a partial accumulation of cells in G1 (12% increase) at 16 h after MIX treatment. This means that the enhanced sensitivity with MIX pretreatment may partly depend on the accumulation of the cells in G1 and which are sensitive to MC. However, other cellular processes may also be involved, since the accumulation in G1 cannot explain all of the sensitization.

Experiments on the quick-relief medical communications via the Japan's domestic communication satellite CS-2 for the case of disasters and emergencies.

Experiments on the quick-relief medical communications via the CS-2 satellite were carried out by using two types of 30/20 GHz small transportable earth stations whose antenna diameters are 1 and 2 m. As the terminal equipments, FM-SCPC systems with a one-telephone-equivalent channel were prepared for the transmission of voice, color freezed picture (9.6 kbps), supersonic echo signal and heart sound from a electrocardiograph. Signals from various medical equipments were transmitted by an FM-SCPC system from Simizu harbour (1 m station) to Tokyo transportable station (2 m), assuming that a person was injured in the ship and the ship came alongside the pier. Transmitted materials are mainly various kinds of pictures of affected parts, X-ray films and electrocardiograph with breathing sounds. It was found possible to send various medical information mentioned above via CS-2 by the 30/20 GHz simple communication systems with one-telephone-equivalent channel. Doctors suggested it would be possible to judge very well about the patients' emergency conditions and to give quick consult with inevitable treatment procedures for them. However, a few problems were found in the Hi-Fi reproduction of original colors and in the transmission of heart sounds in the very low frequency band less than 300 Hz.

[Necessity of co-operation of the community medical system based on the results from the survey by questionnaires-participation of the home healthcare and/or hospice systems in southern Tama community hospitals].

Home Care Division of Fujisawa Pharmaceutical Co. Ltd. has been providing the local public with the following services: 1) providing aseptic medicines prescribed in the clean room, 2) renting the infusion fluid pumps, and 3) supporting the community cooperation in healthcare services. Last year, we surveyed questionnaires to the public users (patients and caretakers) of these services, in order to understand the actual status of patients after changing from conventional hospitalization to the home infusion therapy (HIT). From the results of our present survey, it was found that the patients and their family members had positively accepted HIT, while 61% of the HIT users exhibited a strong anxiety in their skills and methods of HIT. Moreover, it was also shown that 61% had other means of nursing and treatment in addition to HIT, indicating a great financial burden on the families. Among them, 69% of the HIT users considered that visiting nurses and primary care physicians were the best co-operators, and changed their conventional healthcare system (hospitalization) to HIT. However, the home caretakers showed a high anxiety in their skill in the home healthcare system, specifically HIT, which was generally highly dependent on the medical care, Thus, a good relationship and co-operation with visiting nurses and primary care physicians was one of the major factors for the users to decide to choose HIT instead of their old medical hospitalization. Therefore, in order to make HIT more useful and widely prevail, it is concluded that establishment of the co-operative systems within our local community, where visiting nurses and primary care physicians can easily provide the patients and their family with professional suggestions, advice and actual care whenever the home caretakers need them.

The effect of estrogen administration during early pregnancy upon the survival of single implanted pig embryos.

In the present study, we investigated the influence of exogenous estrogen on embryo survival after transfer into prepubertal gilts in which estrus had been induced. In the first experiment, estrus was induced in prepubertal gilts by the administration of 1,000 IU of eCG and 750 IU of hCG every 72 h. Several blastocysts were recovered on d 6 (d 0 is the day of hCG administration), and 1 embryo was transferred to the tip of 1 side of the uterine horn on d 6 (Control). In treated groups, after embryo transfer, 5 mg of estradiol benzoate (EB) was administered on d 11 (EB5mg-1) or d 11, d 13, and d 15 (EB5mg-3) or d 11, 12, 13, 14, and 15 (EB5mg-5) or 20 mg of estradiol dipropionate (EDP) was administered on d 11 (EDP20mg-1) or d 11 and d 14 (EDP20mg-2). Autopsy examinations were performed on d 53 to 60. Although nontreated gilts did not become pregnant, gilts in each of the estradiol-treated groups became pregnant. The greatest pregnancy rate (77.8%, 7/9) was obtained with EDP20mg-2 (EDP20mg-2 > control: P < 0.05). In a second experiment, 1 blastocyst was transferred to prepubertal gilts and treated with EDP20mg-2. Pregnancy in recipient pigs was confirmed by ultrasonography, and pigs were allowed to farrow. Embryo survival rate was high on d 30 of pregnancy (75%, 9/12) but had a tendency (P = 0.0995) to decline from d 30 to delivery (33.3%, 4/12). In a third experiment, prepubertal gilts were administered 5 mg of EDP on d 11 (EDB5mg-1) and d 11 and d 14 (EDP5mg-2). Autopsy examinations were performed on d 53 to 58. Pseudopregnancy rate was high for EDP5mg-2 (63.6%, 7/11) compared with EDP5mg-1 (0%, 0/11; P < 0.05). In a fourth experiment, prepubertal gilts were transferred 1 blastocyst and treated with EDP5mg-2. Pregnancy was confirmed in recipient pigs by ultrasonography, and pigs were subsequently allowed to farrow. Embryo survival rate remained unchanged from d 30 of pregnancy to delivery (66.7%; 8/12). One piglet died from dystocia, and 1 suffered from deformity involving double-breasted hooves and died 6 d after birth. There was no difference (P > 0.05) in survival rate on d 30 of pregnancy and weaning (50%, 6/12). Body weight at birth and at weaning did not differ from that reported in previous studies. In conclusion, this study showed that EDP5mg-2 treatment during early pregnancy leads to full-term development of a single embryo.

Presynaptically silent synapses: spontaneously active terminals without stimulus-evoked release demonstrated in cortical autapses.

This study addresses the question of whether synapses that are capable of releasing transmitters spontaneously can also release them in an excitation-dependent manner. For this purpose, whole cell patch recordings were performed for a total of 48 excitatory solitary neurons in a microisland culture to observe excitatory autaptic currents elicited by spontaneous transmitter release as well as by somatic excitation. A somatic Na+-spike, induced in response to a short voltage step, evoked excitatory postsynaptic currents (EPSCs) of various amplitudes through the autapses; in some cases, no response was noticeable. To make sure that the recorded autaptic spontaneous EPSCs (sEPSCs) under a voltage clamp resulted from independent release of transmitters and were not associated with action potentials, sEPSCS in the presence and absence of tetrodotoxin (TTX) were compared in six cells. In the presence of TTX the evoked EPSCs were completely eliminated, whereas the sEPSCs were still observed and the amplitude distribution histograms were statistically not different from those recorded in the absence of TTX. A quantitative analysis of the sEPSCs (presumably miniature EPSCs) showed that the amplitude of stimulus-evoked EPSCs did not correlate with either the frequency or median amplitudes of the sEPSCs or the age of the culture. To identify whether the absence of stimulus-evoked response was caused either by conduction failure of excitation along the axons or by impairment of the release machinery that links the terminal depolarization to vesicle exocytosis, we examined whether high K+ and hypertonic solutions could facilitate the spontaneous release of transmitters. Although the hypertonic solution increased the spontaneous release in all cells tested (n = 18), the high K+ solution had a differential effect in increasing spontaneous release, i.e., the cells with larger evoked responses were more readily facilitated by the high K+ solution. Because the high K+ solution induced depolarization of presynaptic terminals, the present results indicated that the smaller evoked responses were due to the larger number of impaired or "silent" presynaptic terminals that were unable to link presynaptic depolarization to transmitter release. In summary, the present experiments provided evidence that at least some of the presynaptic terminals are silent in response to stimuli, while remaining spontaneously active at the same time. Because this phenomenon is due to the lack of sensitivity to depolarization at the terminals, these synaptic terminals seem incapable of linking terminal depolarization to transmitter release.

A novel two-site enzyme immunoassay reveals the regional distributions of and developmental changes in GluR1 and NMDAR1 protein contents in the rat brain.

Glutamate receptors, including the alpha-amino-3-hydroxy-4-methylisoxazole-4-propionic acid (AMPA) and NMDA receptors, play an important role in neural development and synaptic plasticity in the brain. To date, it has been difficult to correlate accurately individual biochemical phenomena with quantitative and qualitative changes in receptors occurring in specific neurons or synapses. In the present study, we established a two-site enzyme immunoassay for two key subunits of the AMPA and NMDA receptors. Its sensitivities were extremely high, 30 pg for GluR1 and 15 pg for the NMDAR1 receptor containing the C2 exon [NMDAR1(C2)], which enabled us to measure their contents in a few milligrams of hippocampal tissue. Regional and developmental variations in receptor protein levels were much more marked than those reported for mRNA: The absolute GluR1 protein content was highest in the rat hippocampus, whereas the NMDAR1(C2) content was high in all the forebrain regions examined. GluR1 protein levels increased most markedly during the second and third weeks of postnatal life, whereas NMDAR1(C2) content increased during the first postnatal week. In the adult rat brain, the ratio of GluR1 protein to NMDAR1 protein was markedly lower in neocortical regions (approximately 2%) and the highest in cerebellum (22%). Therefore, this two-site enzyme immunoassay is a specific and unique method that enables us to measure absolute tissue contents of the glutamate receptors and will lead to further important discoveries on the biochemical alterations of these receptors.

Hebbian induction of LTP in visual cortex: perforated patch-clamp study in cultured neurons.

1. To see whether presynaptic activation paired with postsynaptic depolarization is necessary for the induction of long-term potentiation (LTP) in visual cortex or whether an activation of postsynaptic receptors in conjunction with depolarization is sufficient, we carried out perforated patch-clamp recordings with nystatin from cultured cortical neurons of rats. 2. Recorded neurons were monosynaptically activated either by electrical stimulation of an adjacent neuron or by direct activation of glutamate on "hot spots" of dendrites through iontophoresis or pressure ejection. In experiments in which cultured neurons were stained immunocytochemically with antibody against synaptophysin after electrophysiological recordings, hot spots were found to correspond to probable synaptic sites. 3. Excitatory postsynaptic currents (EPSCs) evoked by test stimulation applied to the adjacent neuron at 0.1 Hz were recorded at a holding potential of -60 or -70 mV for 5-10 min after an establishment of the whole cell recording configuration. Then, stimulation was paired with postsynaptic depolarization (0 mV for 200 ms) at 1 Hz for 30 or 60 s. LTP of EPSCs was induced in 7 of the 15 cells from which stable recordings were obtained for 18-30 min after pairing. 4. When postsynaptic depolarization was paired with direct glutamate application in the absence of presynaptic stimulation in 12 cells, only 1 showed LTP. Postsynaptic depolarization alone did not induce LTP in any of the six cells tested. Also, presynaptic stimulation alone did not induce LTP in any of the five cells tested. 5. These results suggest that the concurrent activation of presynaptic elements with postsynaptic depolarization is necessary for the induction of LTP in visual cortex.

Optical recording study of granule cell activities in the hippocampal dentate gyrus of kainate-treated rats.

In the epileptic hippocampus, newly sprouted mossy fibers are considered to form recurrent excitatory connections to granule cells in the dentate gyrus and thereby increase seizure susceptibility. To study the effects of mossy fiber sprouting on neural activity in individual lamellae of the dentate gyrus, we used high-speed optical recording to record signals from voltage-sensitive dye in hippocampal slices prepared from kainate-treated epileptic rats (KA rats). In 14 of 24 slices from KA rats, hilar stimulation evoked a large depolarization in almost the entire molecular layer in which granule cell apical dendrites are located. The signals were identified as postsynaptic responses because of their dependence on extracellular Ca(2+). The depolarization amplitude was largest in the inner molecular layer (the target area of sprouted mossy fibers) and declined with increasing distance from the granule cell layer. In the inner molecular layer, a good correlation was obtained between depolarization size and the density of mossy fiber terminals detected by Timm staining methods. Blockade of GABAergic inhibition by bicuculline enlarged the depolarization in granule cell dendrites. Our data indicate that mossy fiber sprouting results in a large and prolonged synaptic depolarization in an extensive dendritic area and that the enhanced GABAergic inhibition partly masks the synaptic depolarization. However, despite the large dendritic excitation induced by the sprouted mossy fibers, seizure-like activity of granule cells was never observed, even when GABAergic inhibition was blocked. Therefore, mossy fiber sprouting may not play a critical role in epileptogenesis.

Modification of the sensitivity of CHO cells to mitomycin C by dibutyryl cyclic AMP.

The survival of CHO cells exposed to mitomycin C was decreased three times that of the cells treated with 1 mM dibutyryl cyclic AMP before mitomycin C treatment, as compared to the absence of treatment with this cyclic nucleotide. The sensitization effect began at 3-4 hours after the start of pre-treatment, reached a maximum at around 10 hours and continued to be effective. Post-treatment with the cyclic nucleotide for more than 12 hours increased the survival of CHO cells exposed to mitomycin C.

Rapid detection of phylloplane bacterium Enterobacter cloacae based on chitinase gene transformation and lytic infection by specific bacteriophages.

AIMS: To establish a rapid and efficient method for detecting Enterobacter cloacae based on chitinase gene transformation and lytic infection by virulent bacteriophages. METHODS AND RESULTS: A phylloplane strain of E. cloacae was isolated from tomato leaves and transformed with a chitinase gene. Transformed bacteria were collected from single colonies and infected with newly isolated, virulent bacteriophages in the presence of the chitinase substrate 4-methylumbelliferon (4MU)-(GlcNac)3. To assay chitinase activity in the lysates, the product 4MU was measured spectrofluorophotometrically or visibly detected under u.v. irradiation. Chitinase gene-transformed bacteria obtained from single colonies could be specifically identified in 30 min by the emission of 4MU fluorescence following lysis caused by phage infection. CONCLUSIONS: The chitinase gene was used as a reporter gene to construct a new system for easy and rapid monitoring of transgenic strains of E. cloacae released in the environment, in combination with specific recognition by virulent bacteriophages. SIGNIFICANCE AND IMPACT OF THE STUDY: The assay is simple, rapid, inexpensive, easy to perform and applicable to other strains. The system can be used for the routine monitoring of bacteria, which is important because of the increased use of transgenic strains of E. cloacae as an antagonistic biological control agent for plant diseases.

Brain-derived neurotrophic factor upregulates and maintains AMPA receptor currents in neocortical GABAergic neurons.

The regulation of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors is implicated in synaptic plasticity. Although we have found that brain-derived neurotrophic factor (BDNF) triggers surface translocation of AMPA receptor proteins, the physiological significance of the BDNF effect remained to be determined. The present immunohistochemical studies revealed that cortical GABAergic neurons exhibited the most striking response to BDNF. Accordingly, we monitored AMPA-triggered currents through GABAergic neurons: Chronic BDNF treatment increased the AMPA-triggered currents but not NMDA-triggered currents in culture. In parallel, the amplitude, but not frequency, of spontaneous miniature excitatory postsynaptic currents (mEPSCs) was elevated in GABAergic neurons. In agreement, BDNF enhanced GABA release triggered by AMPA compared to the amount triggered by high potassium. Conversely, there was a significant decrease in the mEPSC amplitude of GABAergic neurons in heterozygous BDNF-knockout mice. These findings indicate that the neurotrophin enhances the input sensitivity of GABAergic neurons to facilitate their inhibitory function in the neocortex.