Assay of phospholipase A2 activity of synaptic membranes using a phospholipid transfer protein: stimulation by depolarization.

A phospholipid transfer protein from bovine liver was used to transfer exogenous ([14C]linoleoylphosphatidylethanolamine [14C]linoleoyl PE) into synaptic membranes and synaptosomes without modifying the lipid compositions in order to study the intrinsic activity of phospholipase A2 of the preparations. Results were compared with the conventional method in which the substrate was simply dispersed with the enzyme preparations. Liberation of [14C]linoleic acid from [14C]linoleoyl PE-preloaded synaptic membranes by the transfer protein continued almost linearly over 60 min of incubation in the presence of Ca2+. The dose-response curve of Ca2+ for the activity of phospholipase A2 obtained in the present method was slightly shifted to the left in comparison with the conventional method: Ca2+ even at the concentration of 100 microM significantly enhanced the enzyme activity. Requirement of Ca2+ for the reaction is more specific in the present method than in the conventional one. When synaptosomes were prelabeled with [14C]linoleoyl PE by the transfer protein, the liberation of [14C]linoleic acid during the incubation at 37 degrees C increased linearly over 2 min. The liberation of [14C]linoleic acid was significantly enhanced in the presence of 56 mM KCl, 50 microM veratridine and 50 microM calcium ionophore A23187. These agents did not stimulate the reaction in the absence of Ca2+.

Brain networks affected by synchronized sleep visualized by positron emission tomography.

Nineteen lightly sleep-deprived healthy volunteers were examined with H2(15)O and positron emission tomography (PET). Scanning was performed during wakefulness and after the subjects had fallen asleep. Sleep stage was graded retrospectively from electroencephalogram (EEG) recordings, and scans were divided into two groups: wakefulness or synchronized sleep. Global flow was quantified, revealing no difference between sleep and wakefulness. A pixel-by-pixel-blocked one-way analysis of variance (ANOVA) was performed after correcting for differences in anatomy and global flow. The sum of squares of the z-score distribution showed a highly significant (P < 0.00001) omnibus difference between sleep and wakefulness. The z-score images indicated decreased flow in the thalamus and the frontal and parietal association cortices and increased flow in the cerebellum during sleep. A principal component (PC) analysis was performed on data after correction for global flow and block effects, and a multivariate analysis of variance (MANOVA) on all PC scores revealed significant (P = 0.00004) differences between sleep and wakefulness. Principal component's 2 and 5 correlated to sleep and revealed distinct networks consisting of PC 2, cerebellum and frontal and parietal association cortices, and PC 5, thalamus.

Prostacyclin receptor in the brain and central terminals of the primary sensory neurons: an autoradiographic study using a stable prostacyclin analogue [3H]iloprost.

Presence and localization of the prostacyclin receptor in the rat brain and spinal cord were examined by in vitro autoradiography using [3H]iloprost, a highly specific and stable agonist for this receptor. Density of specific binding sites for iloprost was generally high in four regions of the lower brain stem, that is, the medial and commissural subnuclei of the nucleus tractus solitarius, the area postrema, superficial layers of the spinal trigeminal nucleus caudalis and dorsal horn. Moderate density was found in the thalamus, cerebral cortex, hippocampus, striatum and dorsal cochlear nucleus. The distribution pattern was distinct from those of other prostanoid binding sites previously studied except that prostaglandin E2 binding sites were also abundant in the nucleus tractus solitarius, spinal trigeminal nucleus caudalis and dorsal horn. Even in these regions, binding sites for iloprost had several features clearly different from those for prostaglandin E2. First, within the medial and commissural subnuclei of the nucleus tractus solitarius, iloprost binding sites were distributed preferentially in the dorsal part, while those for prostaglandin E2 were located more ventrolaterally. Second, on postnatal day 0, iloprost binding sites have already been expressed in large amounts in the nucleus tractus solitarius, spinal trigeminal nucleus caudalis and dorsal horn of rats, while prostaglandin E2 binding sites are negligible at this stage. Thirdly, the binding of 10 nM [3H]iloprost in these three regions was almost completely displaced by 10 microM unlabelled iloprost but only slightly by 10 microM unlabelled prostaglandin E2. Unilateral nodose ganglionectomy or dorsal rhizotomy decreased the density of iloprost binding sites in the nucleus tractus solitarius or dorsal horn, respectively, with a greater decrease in the operated side. Ligation of the vagus either central or peripheral to the nodose ganglion resulted in an accumulation of iloprost binding sites proximal to the ligation. These results suggest that specific binding sites for iloprost, presumably prostacyclin receptor, are present in the nervous system and, in particular, that the iloprost binding sites in the nucleus tractus solitarius, dorsal horn and possibly in the superficial layers of the spinal trigeminal nucleus caudalis are produced in their sensory ganglia and transported to central terminals of the primary sensory afferents as well as to their peripheral terminals.

Kainate receptors: a novel mechanism in paradoxical (REM) sleep generation.

Application of kainate to the peri-locus coeruleus alpha (peri-LC alpha) resulted in a marked increase in paradoxical sleep (PS). This effect was completely blocked by specific non-NMDA receptor antagonists, but not by selective NMDA receptor antagonists. Kainate was 10 times more potent than AMPA, and their site of action was confined to the peri-LC alpha. This study is the first to show that excitatory amino acids play an important role in PS generation via the activation of kainate receptors which are located in the peri-LC alpha of the mediodorsal pontine tegmentum.

A subclass of GABAA/benzodiazepine receptor exclusively localized in the limbic system.

An in vivo saturation study using 11C-labelled Ro15-4513 with high specific radioactivity (> 70 GBq mumol-1) revealed the presence of very high-affinity and high-affinity binding sites (Kd values in the amygdala approximately 0.4 and 18.7 nM, respectively) in the gamma-aminobutyric acid type A/benzodiazepine (GABAA/BZ) receptor in the living monkey brain. Subtraction of an image obtained using [11C]Ro15-4513 with low specific radioactivity from an image obtained by that with high specific radioactivity, both of which were scanned with a high spatial resolution positron emission tomography camera, demonstrated that the very high-affinity sites are exclusively localized in the limbic system, such as in the amygdala, hippocampus, anterior cingulate, septum, nucleus accumbens and insular cortex. The localization of the very high-affinity binding sites, which may be derived from a subclass of central GABAA/BZ receptors, might account for the anxiolytic effect of BZ analogues.

Attentional modulation of neural activity in the macaque inferior temporal cortex during global and local processing.

To examine whether visual attention to global and local features of visual stimuli modulates neural activity in the monkey visual cortex, we applied positron emission tomography techniques to monkeys while they were discriminating either global or local features of visual stimuli. The posterior inferior temporal cortex was more activated in discriminating global features than in discriminating local ones, whereas the anterior inferior temporal cortex was more activated in discriminating local features than in discriminating global ones. The results suggest that a functional difference exists in terms of processing of global and local features within the inferior temporal cortex.

Promotion of sleep by prostaglandin D2 in rats made insomniac by pretreatment with para-chlorophenylalanine.

The correlation between the somnogenic effect of prostaglandin (PG) D2 and the serotoninergic system was examined in freely-moving rats (n = 64) by use of a continuous infusion method. Rats pretreated with para-chlorophenylalanine (PCPA: 450 mg/kg body weight, i.p.) or non-PCPA-pretreated rats received infusion of PGD2, serotonin, or its direct precursor, 5-hydroxytryptophan (5HTP), into their third cerebral ventricle at a rate of 100 pmol/0.2 microliter/min between 11:00 and 17:00 h. In the PCPA-pretreated insomniac rats, PGD2 infusion resulted in an immediate increase in slow-wave sleep (SWS) and an increase with a 2-h latency in paradoxical sleep (PS). The total amounts of SWS and PS during the PGD2-infusion period were 151% and 154% of the respective control values. These results indicate that inhibition of the biosynthesis of serotonin and 5HTP by PCPA marginally affects the sleep-promoting effect of PGD2. The transient sleep restoration produced by 5HTP infusion into PCPA-pretreated rats was hardly affected by the simultaneous infusion (200 pmol/0.2 microliter/min; 07:00-17:00 h) of diclofenac sodium, an inhibitor of cyclo-oxygenase, suggesting that PGD2 production is not critically involved in the sleep restoration by 5HTP. The sleep-promoting property of PGD2 is thus probably independent of the serotoninergic modulation of sleep-wake activity.

A novel near infra-red spectrophotometry system using microprobes: its evaluation and application for monitoring neuronal activity in the visual cortex.

A novel near-infrared (NIR) spectrophotometry system with microprobes of optical transmitter and receivers (550 and 410 microm in diameter, respectively) has been developed. A three-dimensional profile of the signal source estimated in in vitro experiments showed two spindle-shaped regions around the respective probes, suggesting that the signal detected by the present system comes from a relatively restricted region around each probe. Next, we examined how the concentration of oxygen in inspired gas affected the NIR signals in the rat cerebral cortex in vivo. Calculated concentrations of oxyhemoglobin (HbO2) and deoxyhemoglobin (Hb) in the rat cortex changed in mirror-image fashion in response to the change in the O2 concentration in the inspired gas. Finally, NIR responses to visual stimulation were recorded from the striate cortex of conscious adult cats that had been monocularly deprived since the peak of the sensitive period. Corresponding to the results of previous electrophysiological studies, stimulation of the normal eye induced significant NIR signals, whereas that of the deprived eye evoked no response. These results indicate that this new NIR system can be applied to study changes in oxygen metabolism in relatively restricted regions following neuronal activation in the brain.

In vitro positron emission tomography (PET): use of positron emission tracers in functional imaging in living brain slices.

Positron-emitting radionuclides have short half-lives and high radiation energies compared with radioisotopes generally used in biomedical research. We examined the possibility of applying positron emitter-labeled compounds to functional imaging in brain slices kept viable in an oxygenated buffer solution. Brain slices (300 microns thick) containing the striatum were incubated with positron emitter-labeled tracers for 30-45 min. The slices were then rinsed and placed on the bottom of a Plexiglas chamber filled with oxygenated Krebs-Ringer solution. The bottom of the chamber consisted of a thin polypropylene film to allow good penetration of beta+ particles from the brain slices. The chamber was placed on a storage phosphor screen, which has a higher sensitivity and a wider dynamic range than X-ray films. After an exposure period of 15-60 min, the screen was scanned by the analyzer and radioactivity images of brain slices were obtained within 20 min. We succeeded in obtaining quantitative images of (1) [18F]fluorodeoxyglucose uptake, (2) dopamine D2 receptor binding, (3) dopa-decarboxylase activity, and (4) release of [11C]dopamine preloaded as L-[11C]DOPA in the brain slice preparation. These results demonstrate that positron emitter-labeled tracers in combination with storage phosphor screens are useful for functional imaging of living brain slices as a novel neuroscience technique.

Receptor imaging technique with 11C-labeled receptor ligands in living brain slices: its application to time-resolved imaging and saturation analysis of benzodiazepine receptor using [11C]Ro15-1788.

Recently we developed a novel imaging technique using positron emitter-labeled compounds as probes and a storage phosphor screen as a detector. This approach makes it possible to follow a variety of biochemical processes with spatial information in living brain slices. Further technical development is reported here in terms of time-resolved imaging and receptor characterization in a real equilibrium state. The method was validated by use of [11C]Ro15-1788, a benzodiazepine receptor antagonist. Fresh brain slices were incubated with [11C]Ro15-1788 in oxygenated Krebs-Ringer solution at 37 degrees C, in a specially designed chamber. By placing the chamber on a storage phosphor screen, we could obtain two-dimensional images of radioactivity in the slices. Time-resolved imaging was made at 5 min intervals, revealing that it took 60 min to reach equilibrium binding. The dissociation process was observed by adding an excess amount of unlabeled Ro15-1788 to the chamber, 25 min was required for the full dissociation. In the equilibrium state, i.e. in the presence of free radio-ligand, Scatchard plot analysis was performed on the cerebral cortex (Kd = 7.4 nM, Bmax = 146 fmol/mg tissue) and striatum (Kd = 7.5 nM, Bmax = 107 fmol/mg tissue), suggesting the presence of a single component of binding site in these two regions. The present method, for the first time, made it possible to study a ligand-receptor interaction in living brain slices with temporal and spatial resolutions. This technique should prove useful for studies of receptor function under physiological conditions.

GABAergic system inducing hyperthermia in the rat preoptic area: its independence of prostaglandin E2 system.

Brain temperature of conscious freely moving rats was recorded during perfusion of the preoptic area (POA) with neuroactive compounds using the microdialysis technique. Unilateral perfusion of the POA with the sodium channel blocking agent, tetrodotoxin (1 microM), induced a pronounced hyperthermia. Of the neuroactive compounds examined, the greatest thermogenic response to local perfusion of the POA was elicited by the GABAergic agonist, muscimol. Muscimol (10, 20 and 100 microM) exhibited a dose-dependent and reversible hyperthermia. This hyperthermia was attenuated by co-perfusion with the GABAergic antagonist, bicuculline (10 microM). Muscimol-induced hyperthermia was independent of prostaglandin biosynthesis, and additive with prostaglandin E2 (10 microM)-induced hyperthermia. Prostaglandin E2-induced hyperthermia was not affected by co-perfusion with bicuculline. These data suggest the existence of two independent neurochemical systems for genesis of hyperthermia colocalized within the POA.

High density of prostaglandin E2 binding sites in the anterior wall of the 3rd ventricle: a possible site of its hyperthermic action.

Prostaglandin E2 (PGE2) exerts a potent hyperthermic action when injected into the preoptic-hypothalamic area (POHA) and is considered to be a central mediator of fever. To determine the exact functional sites of PGE2, we used in vitro quantitative autoradiography of [3H]PGE2 binding sites in the rat POHA. The highest density of [3H]PGE2 binding was found in the regions of the anterior wall of the 3rd ventricle (A3V). Within the A3V, binding density was especially high in regions closest to the third ventricle or surrounding the organum vasculosum laminae terminalis (OVLT) but was relatively low within the OVLT itself. It seems likely that the A3V PGE2 binding sites identified in this study are responsible for PGE2 mediation of fever.

Mapping of prostaglandin E2 binding sites in rat brain using quantitative autoradiography.

The density of specific prostaglandin E2 (PGE2) binding sites was quantitatively mapped in the rat brain using in vitro autoradiography. The anterior wall of the third ventricle and the nucleus solitary tract were found to have a very high density of binding sites (greater than 15 fmol/mg tissue). Two thalamic nuclei (paraventricular and anteroventral nuclei) and the dorsal parabrachial nucleus contained a high density of binding sites (10-15 fmol/mg tissue). Entorhinal cortex, ventral hippocampus, amygdala, dorsomedial hypothalamus, mammillary complex, some thalamic nuclei, central gray, superior colliculus, raphe nuclei, locus coeruleus, spinal trigeminal nucleus (caudal part) and the dorsal horn of the spinal cord (laminae 1 and 2) had each a moderate density of binding sites (5-10 fmol/mg tissue). Binding tended to occur in brain regions rich in neuronal cell bodies or neuronal cell processes (dendrites and axon terminals). PGE1, whose central actions are very similar to those of PGE2, had essentially the same pattern of binding sites as did PGE2 throughout the entire brain, suggesting there are receptors common to these two PGEs. In addition to already known functions of receptors common to these two PGEs. In addition to already known functions of PGE2 in the hypothalamus, which include fever genesis, promotion of wakefulness, cardiovascular control and LH-RH release, the unique distribution of extrahypothalamic PGE2 binding sites found in this study suggests its involvement in the processing or modulation of viscerosensory, somatosensory (nociceptive and possibly thermal) and visual inputs as well as in the central integration of autonomic and limbic functions.(ABSTRACT TRUNCATED AT 250 WORDS)

Prostaglandin E2 excites neurons of the nucleus tractus solitarius by activating cation channels.

Nucleus tractus solitarius (NTS) has a high density of prostaglandin E2 (PGE2)-binding sites. Action of PGE2 (10(-9)-10(-6) M) was tested on neurons in a NTS slice with patch-clamp recording under synaptic blockade. PGE2 raised the firing rate in approximately half of the neurons in cell-attached patch mode. In whole-cell current clamp, PGE2 depolarized membrane potential accompanied by an increase in firing rate. In whole-cell voltage clamp (-58 mV), PGE2 induced the inward current with an increase in conductance. The current was linearly related to voltage from -100 mV to -10 mV and suppressed between -10 mV and 20 mV. The current-voltage curve remained similar under low external Cl- or high internal Cl- conditions and after external Na+ was replaced by Cs+. It is concluded that PGE2 excites NTS neurons by activating cation conductance.

Ketamine increases the striatal N-[11C]methylspiperone binding in vivo: positron emission tomography study using conscious rhesus monkey.

A system for positron emission tomography study of conscious monkeys was newly developed. By use of this system in combination with a microdialysis technique, the effect of ketamine on the binding and release of dopamine was investigated. The administration of ketamine (5 mg/kg) caused sedation accompanied by psychotic symptoms such as nystagmus and stereotyped movements of extremities. During this psychotomimetic period produced by ketamine, a significant increase in the accumulation of the dopamine D2 receptor ligand N-[11C]methylspiperone was observed in the striatum compared with the level in the conscious state, while no significant change was observed in the frontal cortex and cerebellum. In contrast to the use of ketamine as the anesthetic, pentobarbital (25 mg/kg), which produced deeper anesthesia but no psychotic symptoms, caused a decrease in the accumulation of N-[11C]methylspiperone in the striatum. Kinetic analysis, conducted by a graphical method, revealed that the value of the association constant (K3) for N-[11C]methylspiperone binding in the striatum was increased to approximately 130% by ketamine and decreased to approximately 70% by pentobarbital compared with the control values. Furthermore, the release of dopamine from the striatum measured by microdialysis was not affected by ketamine anesthesia. These results indicate that ketamine facilitates striatal dopaminergic neurotransmission through increasing the binding activity of dopamine D2 receptors in the striatum, and suggest that these changes may be related to the psychotomimetic behavioral symptoms of this drug.

REM sleep-associated hemoglobin oxygenation in the monkey forebrain studied using near-infrared spectrophotometry.

The oxygenation state of hemoglobin (Hb) in the monkey forebrain was monitored continuously throughout nocturnal sleep using a near-infrared spectrophotometric technique. During rapid eye movement (REM) sleep, an increase in oxygenated Hb occurred concomitantly with a decrease in deoxygenated Hb, while no significant changes in Hb oxygenation were observed during slow wave sleep (SWS). A gradual increase in total Hb content was also observed to occur during each REM sleep episode but not during SWS.

Behavioral activation by stimulation of a GABAergic mechanism in the preoptic area of rat.

The locomotor activity and grooming of conscious freely moving rats were recorded during a 60-min unilateral perfusion of the preoptic area with neuroactive compounds using the microdialysis technique. The GABA agonist, muscimol (10, 20 and 100 microM) induced a dose-dependent increase in locomotor activity and grooming which was attenuated by co-perfusion with the GABA antagonist, bicuculline (10 microM), and was blocked by systemic injection of haloperidol, a preferential dopamine D2 receptor antagonist (0.25 mg/kg). Muscimol-induced hyperactivity was associated with a simultaneous increase of striatal extracellular dopamine. These data suggest that the preoptic area is functionally linked with the extrapyramidal dopaminergic system possibly via GABAergic system.

Non-synchronous behavior of neuronal activity, oxidative metabolism and blood supply during mental tasks in man.

In near-infrared spectroscopic studies during mental tasks such as problem solving and mental arithmetic, we found that 9 of 33 healthy volunteers showed decreases in both the regional cerebral blood flow (r-CBF) and oxygen consumption rate (CMRO2) in the frontal region of the dominant hemisphere. To confirm these unexpected observations, we performed simultaneous measurements by positron emission tomography (PET) and near-infrared spectroscopy (NIRS) in two such subjects. PET images also showed that CBF decreased within the presumptive area illuminated by near-infrared light during mental task. However, CBF decreased in almost all regions while the subject gave a correct answer. Thus, the questions arose: Are mental tasks always associated with increases in r-CBF and/or CMRO2?

Mapping of cortical areas involved in color vision in non-human primates.

Positron emission tomography (PET) was used to measure changes in the regional cerebral blood flow (rCBF) of rhesus monkeys performing visual discrimination tasks. In comparison with both position and brightness discrimination tasks, the color discrimination task activated the posterior inferior temporal cortex and a ventromedial occipital region, which is located along the anterior one-third of the calcarine sulcus. In contrast, the position task activated the middle temporal area and intraparietal cortex as compared with the color task. These results confirm the segregation of visual pathways and delineate the visual areas involved in color vision. This approach might bridge the gap between invasive studies in animals and functional imaging studies in humans.

Brain activation study by use of positron emission tomography in unanesthetized monkeys.

A system for the measurement of brain activity in conscious monkeys by positron emission tomography (PET) was established in the present study. The signal/noise ratio was maximal around 40 s for data acquisition in the PET scan with 15O-labeled water. When the monkey was stimulated by vibration and subtraction images of the data sets from regional cerebral blood flow (rCBF) changes in paired stimulation and control were superimposed on magnetic resonance images obtained from the same specimens, a somatotopic map corresponding to the sites stimulated was clearly demonstrated. Visual stimulation with a photic stimulator activated the corresponding regions of the primary visual cortex. Comparison of the activated sites and extents under the conscious state with those under anesthesia assured that the study is controllable; there was little unpredictable activation due to unlimited subject movement or to psychological effects.