Alterations of 45Ca accumulation and [3H]inositol 1,4,5-trisphosphate binding using autoradiography in the exo-focal postischemic brain areas of the rat.

We studied the alterations of calcium accumulation and intracellular signal transduction using autoradiography of the second messenger system in order to clarify the mechanisms of the delayed neuronal changes in the remote areas of rat brain after transient focal ischemia. Chronological changes of 45Ca accumulation and [3H]inositol 1,4,5-trisphosphate (IP3) binding sites were determined after 90 min of right middle cerebral artery (MCA) occlusion and after such occlusion followed by different periods of recirculation. After the ischemic insult, 45Ca accumulation extended to the lateral segment of the caudate putamen and to the cerebral cortex, both supplied by the occluded MCA. One day after the ischemia, [3H]IP3 binding sites decreased significantly compared with the control values in these ischemic areas. Moreover, 3 days after the ischemia, 45Ca accumulation was first detected in the ipsilateral thalamus and the substantia nigra, which lay outside the ischemic areas. In the substantia nigra, a significant decrease of [3H]IP3 binding sites and concurrent 45Ca accumulation were observed. In the thalamus, however, there was not alteration until 1 week after the ischemia, and then [3H]IP3 binding sites increased significantly 2 weeks (P less than 0.05) and 4 weeks (P less than 0.01) after the ischemia. Based on the present study, we speculate that different mechanisms associated with signal transduction systems may be responsible for exo-focal postischemic delayed neuronal changes in the thalamus and the substantia nigra. The increase of [3H]IP3 binding sites of the thalamus in the chronic stage may be new evidence of plasticity related to neurotransmission.     

Alteration of protein kinase C activity in the postischemic rat brain areas using in vitro [3H]phorbol 12,13-dibutyrate autoradiography

Summary Chronological changes of protein kinase C (PKC) activity were measured using in vitro [³H]phorbol 12,13-dibutyrate (PDBu) autoradiography to investigate the postischemic alteration of this second messenger system in the rat brain. Transient ischemia was induced by the occlusion of the middle cerebral artery (MCA) for 90 min and such occlusion followed by various recirculation periods of up to 4 weeks. After 90 min of ischemia followed by 3 hours of recirculation, [³H]PDBu binding sites were found to be significantly decreased in the cerebral cortex and lateral segment of the caudate putamen, both supplied by the occluded MCA; thereafter, the binding sites decreased progressively in those ischemic foci. On the contrary, there was no alteration on day 1, but 3 days after ischemic insult, a significant decrease of [³H]PDBu binding sites was first detected in the ipsilateral thalamus and the substantia nigra, which both areas had not been directly affected by the original ischemic insult. This postischemic delayed phenomenon observed in the thalamus and the substantia nigra developed concurrently with⁴⁵Ca accumulation, which was detected there in our previous study. These results suggest that alteration of second messenger (PKC) pathways may be involved not only in the ischemic foci, but also in neuronal degeneration of the exo-focal remote areas in relation to the disruption of intracellular calcium homeostasis which plays a key role in the pathogenesis of postischemic neuronal damage and that marked alteration of intracellular signal transduction may precede the neuronal damage in the exo-focal postischemic brain areas.     

Exo-focal postischemic neuronal damage in the rat brain: alteration of [H]forskolin binding using in vitro autoradiography

We studied the alteration of intracellular signal transduction using quantitative autoradiography of the second messenger system in order to clarify the mechanisms of delayed neuronal damage in the remote areas of rat brain after transient focal ischemia. Chronological changes of [³H]forskolin binding sites were measured to demonstrate the striatal-nigral pathway after 90 min of right middle cerebral artery (MCA) occlusion and after such occlusion followed by 3 h, 6 h, 1 day, 3 days, 1 week, 2 weeks and 4 weeks of recirculation. [³H]Forskolin binding sites were found to be markedly decreased in the lateral segment of the caudate putamen supplied by the occluded MCA after 90 min of ischemia with no recirculation. On the contrary, there was no alteration on day 1, but 3 days after ischemic insult, marked reduction of [³H]forskolin binding sites was observed in the ipsilateral substantia nigra which lay outside the ischemic areas. This postischemic delayed phenomenon observed in the substantia nigra developed concurrently with ⁴⁵Ca accumulation, which was detected there in our previous study. The delayed reduction of [³H]forskolin binding sites in the substantia nigra observed in the present study indicates that striatonigral terminal degeneration at presynaptic sites is caused by precedent ischemic damage of the ipsilateral caudate putamen and that exo-focal postischemic neuronal death is caused by a transsynaptic process associated with the ischemic foci.     

Age-dependent changes in second messenger and rolipram receptor systems in the gerbil brain

Summary Age-related alterations in binding sites of major second messengers and a selective adenosine 3,5-cyclic monophosphate (cyclic-AMP) phospho-diesterase (PDE) in the gerbil brain were analysed by receptor autoradiography. [³H]Phorbol 12,13-dibutyrate (PDBu), [³H]inositol 1,4,5-trisphosphate (IP₃), [³H]forskolin, [³H]cyclic-AMP, and [³H]rolipram were used to label protein kinase C (PKC), IP₃ receptor, adenylate cyclase, cyclic-AMP dependent protein kinase (PKA), and Ca²⁺/calmodulin-mdependent cyclic-AMP PDE, respectively. In middle-aged gerbils (16 months old), [³H]PDBu binding was significantly reduced in the hippocampal CA 1 sector, thalamus, substantia nigra, and cerebellum, compared with young animals (1 month old). [³H]IP₃ binding revealed significant elevations in the nucleus accumbens, hippocampal CA 1 sector, dentate gyrus, and a significant reduction in cerebellum of middle-aged gerbils. [³H]Forskolin binding in middle-aged animals was significantly increased in the nucleus accumbens and hilus of dentate gyrus, but was diminished in the substantia nigra and cerebellum. On the other hand, in middle-aged animals, [³H]cyclic-AMP binding revealed a significant elevation only in the hippocampal CA3 sector, whereas [³H] rolipram binding showed a significant reduction in the thalamus and cerebellum. Thus, the age-related alteration in these binding sites showed different patterns among various brain regions in middle-aged gerbils indicating that the binding sites of PKC, IP₃, and adenylate cyclase are more markedly affected by aging than those of PKA and cyclicAMP PDE and that the hippocampus and cerebellum are more susceptible to these aging processes than other brain regions. The findings suggest that in-tracellular signal transduction is affected at an early stage of senescence and this may lead to neurological deficits.     

Age-related changes in bindings of second messengers in the rat brain

Age-related alterations in bindings of major second messengers in the brain were studied in 3-week- and 6-, 12-, 18- and 24-month-old Fisher 344 rats using receptor autoradiography. [³H]Phorbol 12,13-dibutyrate (PDBu) and [3H]forskolin were used to label protein kinase C (PKC) and adenylate cyclase, respectively. In immature rats (3-week-old), [³H]PDBu binding showed a significant decrease only in the cerebellum as compared to adult rats (6-month-old), whereas [³H]forskolin binding exhibited a significant reduction in the neocortex, nucleus accumbens, thalamus and substantia nigra. In aged rats, [³H]PDBu binding showed no significant change in all brain areas. In contrast, [³H]forskolin binding showed a conspicuous reduction in various brain areas in 18-month-old rats as compared to adult animals. The age-related reduction was especially observed in the cerebral cortex, hippocampal CA3 pyramidal cell layer, dentate gyrus, thalamus and molecular layer of cerebellum of 24-month-old rats. The results indicate that adenylate cyclase system in the rat brain is more susceptible to aging processes than phosphoinositide cycle system. Furthermore, our data demonstrate that the change in the adenylate cyclase system is more pronounced than that in the phosphoinositide cycle system in immature rat brain. These findings suggest that the adenylate cyclase system is primarily affected in aging processes and this may lead to age-related neurological deficits.     

Immunocytochemical localization of DARPP-32, a dopamine and cyclic-AMP-regulated phosphoprotein, in the primate brain.

The localization of DARPP-32, a dopamine and cAMP-regulated phosphoprotein, has been studied in monkey brain by immunocytochemistry. This study indicates that DARPP-32 is enriched in neurons in regions receiving a dense dopamine input from the substantia nigra and ventral tegmental area. Thus, the majority of somata in the anterior olfactory area, nucleus accumbens, caudate nucleus, and putamen are immunoreactive for DARPP-32. In the caudate nucleus, immunoreactive spines receive asymmetric contacts from unlabeled axon terminals. Immunoreactive somata have diameters of 10-15 microns. In regions known to receive projections from these nuclei, immunoreactivity is confined to small puncta that represent axons and axon terminals. Regions in which immunoreactivity is present in puncta include the ventral pallidum, globus pallidus, and substantia nigra pars reticulata. Dopaminergic neurons themselves are not immunoreactive. Neurons containing moderate to weak immunoreactivity for DARPP-32 are observed in portions of the cerebral cortex, particularly in the temporal cortex (layer VI). DARPP-32-positive neurons are also present in the cerebellum, in the medial habenula, and in portions of the bed nucleus of the stria terminalis and amygdaloid complex. DARPP-32 immunoreactivity is also present in astrocytes in the subcortical white matter and in tanycytes in the arcuate nucleus and median eminence. DARPP-32 may be an effective marker for dopaminoceptive neurons in which the actions of dopamine on the D-1 dopamine receptor are mediated through cAMP and its associated protein kinase.     

Autoradiographic analysis of second messenger and neurotransmitter system receptors in the gerbil hippocampus following transient forebrain ischemia.

Changes in second messenger and neurotransmitter system receptor ligand binding induced by transient forebrain ischemia were studied in the gerbil hippocampus. The animals were allowed variable periods of recovery ranging from 2 h to 7 days after 5-min bilateral carotid artery occlusion. The binding of second messenger systems ([3H]inositol 1,4,5-trisphosphate ([3H]IP3)to inositol 1,4,5-triphosphate, [3H]forskolin to adenylate cyclase and [3H]phorbol 12,13-dibutylate to protein kinase C) and neurotransmitter receptor systems ([3H]PN200-110 to L-type calcium channels. [3H]N6-cyclohexyl-adenosine to adenosine A1 and [3H]quinuclidinyl benzilate to muscarinic cholinergic receptor) were assayed using quantitative autoradiography. In the CA1 subfield, 2 h after ischemia, [3H]IP3, [3H]forskolin, and [3H]quinuclidinyl benzilate binding activities significantly decreased by 25, 17 and 13%, respectively, though no morphological abnormalities were obvious. Six hours after ischemia, the [3H]phorbol 12,13-dibutylate binding activity in the stratum oriens of the CA1 subfield increased by 15%. One day after ischemia, [3H]PN200-110 binding activity in this subfield decreased by 26%, and 7 days after ischemia, [3H]phorbol 12,13-dibutylate and [3H]N6-cyclohexyl-adenosine receptor binding activities decreased in this subfield. In particular, at 7 days after ischemia, [3H]IP3 binding activity in the CA1 subfield showed a complete decline. In the CA3 subfield, [3H]PN200-110 binding activity decreased 2 days after ischemia, and [3H]IP3 and [3H]N6-cyclohexyl-adenosine binding activities decreased 7 days after ischemia. In the dentate gyrus, the structure of which remained histologically intact after ischemic insult, [3H]IP3 and [3H]forskolin binding activities decreased 7 days after ischemia. In contrast, the [3H]phorbol 12,13-dibutylate binding activity increased in the molecular layer of the dentate gyrus 7 days after ischemia. These results indicate that marked alteration of intracellular signal transduction precedes neuronal damage in the hippocampal CA1 subfield and that the histologically intact CA3 and dentate gyrus also shows modulated neuronal transmission after ischemia.     

Distribution of D2 dopamine receptor mRNA in the primate brain.

1. The distribution of the messenger RNA (mRNA) encoding the D₂dopamine receptor has been mapped in the monkey brain by hybridization.

2. Using [35s]-labelled riboprobes corresponding to the region of the D2 dopamine receptor spanning the third cytosolic loop and the sixth and seventh transmembrane domains, specific hybridization was observed in a number of neural structures.

3. High levels of mRNA expression were observed in the caudate, putamen, and claustrum. Significant amounts were also identified in the hippocampus, lateral geniculate nucleus, much of the cortex, amygdala, pons, and thalamus. High levels of this mRNA were also visualized in the substantia nigra, likely reflecting autoreceptor synthesis.

4. While the distribution of D₂ dopamine receptor mRNA was similar between the monkey and previously published maps in the rat, several differences were noted.

5. These results demonstrate the feasibility of visualizing this mRNA in the primate brain, and suggest that a similar analysis of human postmortem brain material may be possible.     

Polyamine metabolism and glutamate receptor agonists-mediated excitotoxicity in the rat brain.

Putrescine (PUT) increases have been seen in a range of models of neuropathological disturbances. The present study was designed to compare the ability of various types of glutamate receptor agonist to promote excitotoxic brain damage and to examine whether a PUT increase is a general marker of excitotoxic brain damage. To that end, we evaluated features of brain damage associated with the excitotoxicity induced by both ionotropic glutamate receptor (iGluR) and metabotropic glutamate receptor (mGluR) agonists in the conscious rat and the changes produced in the regulation of polyamine metabolism. Intracerebroventricular infusion of N-methyl-D-aspartate (NMDA; 80 nmol), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA; 15 nmol), kainic acid (KA; 2.3 nmol), (R,S)-3,5-dihydroxyphenylglycine (3,5-DHPG; 1.5 micromol), and (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD; 2 micromol) produced similar seizure incidences (76-84%) in the rat. The convulsant episodes appeared sooner after iGluR (13-22 min) than after mGluR agonists (50-179 min). Histological analysis of the hippocampus 24 hr after seizures indicated several degrees of excitotoxic injury after equiconvulsive doses of the iGluR and mGluR agonists assayed. The agonists can be placed in the following order, according to the degree of damage they produce: AMPA > 3,5-DHPG approximately KA > NMDA > 1S,3R-ACPD. In the frontal cortex, moderate to low levels of damage were observed after all GluR agonists. Both iGluR- and mGluR-induced seizures produced an overshoot in the hippocampal and cortical PUT concentration, whereas spermidine and spermine levels were similar to control. Moreover, a concurrence of increased PUT levels and brain damage was observed, indicating that PUT is a general marker of excitotoxic brain damage.     

Alterations in the adenylate cyclase-cAMP-phosphodiesterase system in the locus coeruleus during the development of spontaneous hypertension of the rat

The quantitatively most important noradrenergic cell group of the brain is the locus coeruleus. Significantly increased cAMP concentration could be measured in spontaneously hypertensive rats in comparison to normotensive Wistar-Kyoto control rats at every stage investigated. Furthermore, both the basal activity and maximal stimulation of Ca++- and GTP-dependent adenylate cyclase as well as phosphodiesterase activity were significantly decreased in the spontaneously hypertensive rats at 14 weeks of age. The possible role of the locus coeruleus in spontaneous hypertension is presumed in counterregulatory mechanisms against rising blood pressure.     

Distribution of pre-pro-glucagon and glucagon-like peptide-1 receptor messenger RNAs in the rat central nervous system

Glucagon-like peptide-1 (GLP-1) is derived from the peptide precursor pre-pro-glucagon (PPG) by enzymatic cleavage and acts via its receptor, glucagon-like peptide-1 receptor (GLP-1R). By using riboprobes complementary to PPG and GLP-1R, we described the distribution of PPG and GLP-1R messenger RNAs (mRNAs) in the central nervous system of the rat. PPG mRNA-expressing perikarya were restricted to the nucleus of the solitary tact or to the dorsal and ventral medulla and olfactory bulb. GLP-1R mRNA was detected in numerous brain regions, including the mitral cell layer of the olfactory bulb; temporal cortex; caudal hippocampus; lateral septum; amygdala; nucleus accumbens; ventral pallium; nucleus basalis Meynert; bed nucleus of the stria terminalis; preoptic area; paraventricular, supraoptic, arcuate, and dorsomedial nuclei of the hypothalamus; lateral habenula; zona incerta; substantia innominata; posterior thalamic nuclei; ventral tegmental area; dorsal tegmental, posterodorsal tegmental, and interpeduncular nuclei; substantia nigra, central gray; raphe nuclei; parabrachial nuclei; locus ceruleus, nucleus of the solitary tract; area postrema; dorsal nucleus of the vagus; lateral reticular nucleus; and spinal cord. These studies, in addition to describing the sites of GLP-1 and GLP-1R synthesis, suggest that the efferent connections from the nucleus of the solitary tract are more widespread than previously reported. Although the current role of GLP-1 in regulating neuronal physiology is not known, these studies provide detailed information about the sites of GLP-1 synthesis and potential sites of action, an important first step in evaluating the function of GLP-1 in the brain. The widespread distribution of GLP-1R mRNA-containing cells strongly suggests that GLP-1 not only functions as a satiety factor but also acts as a neurotransmitter or neuromodulator in anatomically and functionally distinct areas of the central nervous system. J. Comp. Neurol. 403:261–280, 1999. © 1999 Wiley-Liss, Inc.     

Osteopontin: correlation with phagocytosis by brain macrophages in a rat model of stroke

Osteopontin (OPN) is an adhesive glycoprotein linked to a variety of pathophysiological processes. We investigated whether OPN might act as an opsonin in the diseased brain by studying the postischemic expression and localization of OPN mRNA and protein in a rat model of ischemic stroke. In addition, we characterized the subcellular localization of OPN protein in the ischemic brain core. Induction of OPN mRNA occurred in activated microglia/macrophages in the ischemic core on days 3-7 after reperfusion and this was sustained up to day 28, at least. OPN protein was synthesized and secreted by brain macrophages, which first surrounded damaged striatal white matter tracts and then infiltrated into them. Punctate OPN-immunoreactive profiles were scattered throughout the infarction core except in white matter bundles. Electron microscopy showed the localization of OPN protein along the membranes lining what appeared to be the debris of dead neurons. These were located in the extracellular space and within the cytoplasm of brain macrophages, indicating that the OPN protein accumulated selectively on the surface of dead cells, most of which were phagocytosed subsequently by brain macrophages. However, no significant induction of OPN occurred in degenerating striatal white matter tracts or in brain macrophage-engulfed axonic or myelin debris. These data suggest that OPN secreted by brain macrophages in this rat model of stroke might be involved in the phagocytosis of fragmented cell debris and possibly not in the phagocytosis of axonic or myelin debris.     

Effect of brain microdialysis on aminergic transmitter levels in repeated cerebral global transient ischemia in rat

The effect of repeated transient global ischemia and microdialysis on changes in aminergic neurotransmitter release was investigated using the rat four-vessel occlusion model of global ischemia. To examine the possible transient or permanent changes in neurotransmitter release, ischemia was induced at varying time points in 5 groups of rats. The first ischemia occurred either 24 h (groups I, II, IV, V) or 96 h (group III) following vertebral artery electro-coagulation and guide probe implantation(s), and the second ischemia was induced either 48 h (groups I, IV, V) or 72 h (group II) following the first ischemia. To assess the consequence of repeated microdialysis on the results, one group of rats (group IV) was not dialysed during the first ischemia and another group (group V) was bilaterally dialysed during the second ischemia. Finally, amphetamine-induced neurotransmitter release was also studied in rats submitted to ischemia and compared with that in normal rats. In each case, dopamine, serotonin and their main metabolites were measured by HPLC with electrochemical detection. Monoamine release was inhibited during the second episode of transient ischemia; this non-release was linked to the repeated microdialysis and not to the repeated ischemia. Although the results of chronic studies using brain microdialysis have been widely recognized as valid, the findings presented here indicate that combined with ischemia, probe reinsertion modifies the level of neurotransmitter release.     

Autoradiographic distribution of serotonin transporters and receptor subtypes in human brain

Several neurochemical in vitro and in vivo imaging studies have been aimed at characterizing the localization of serotonin receptors and transporters in the human brain. In this study, a detailed comparison of the distribution of a number of 5-HT receptor subtypes and the 5-HT transporter was carried out in vitro using human postmortem brain tissue. Anatomically adjacent whole hemisphere sections were incubated with specific radioligands for the 5-HT(1A), 5-HT(1B), 5-HT(2A), 5-HT(4) receptors and the 5-HT transporter. The autoradiograms revealed different laminar and regional distribution patterns in the isocortex, where 5-HT(1A) and 5-HT(4) receptor binding showed highest densities in superficial layers and 5-HT(2A) receptor binding was most abundant in middle layers. In cortical regions, 5-HT transporters were concentrated to several limbic lobe structures (posterior uncus, entorhinal, cingulate, insular and temporal polar regions). 5-HT(1A) receptor densities were also high in limbic cortical regions (hippocampus, posterior entorhinal cortex, and subcallosal area) compared to the isocortex. Subregionally different distribution patterns were observed in the basal ganglia with a trend toward higher levels in ventral striatal (5-HT(1B) receptors) and pallidal (5-HT transporters and 5-HT(1B) receptors) regions. The localization in regions belonging to limbic cortico-striato-pallido-thalamic circuits is in line with the documented role of 5-HT in modulation of mood and emotion, and the suggested involvement of this system in pathophysiology of various psychiatric disorders. The qualitative and quantitative information reported in this study might provide important complements to in vivo neuroimaging studies of the 5-HT system.     

白细胞介素1受体拮抗剂对大鼠外伤性脑水肿的治疗作用

目的 观察白细胞介素1受体拮抗剂(IL—1ra)对于外伤性脑水肿的影响。方法 液压伤致大鼠外伤性脑水肿。伤后脑室注射IL-1ra。伤后24h磁共振，脑组织干湿重，HE病理图像分析及电镜等方法观察鼠脑水肿情况。结果 经IL-1ra治疗，与创伤组或治疗对照组相比在磁共振T2加权像上可以水肿减轻；创伤部位脑含水量减低；病理切片可见相同的变化；超微病理发现IL—1ra具有保护作用。结论 IL-1ra对于外伤性脑水肿有治疗作用，内源性IL-1参与了外伤性脑水肿的发生。     

A qualitative comparison of the glucocorticoid receptor in cytosol from human brain and rat brain

The glucocorticoid receptor in cytosol from human brain was studied using isoelectric focussing in slabs of polyacrylamide gel. [³H]Dexamethasone was used as tracer for receptor analysis. The glucocorticoid receptor from human brain was compared to the glucocorticoid receptor in rat brain. A similar peak of radioactivity with a pI of about 6.1 was obtained by isoelectric focussing of cytosol from both human brain and rat brain.The trypsin-induced fragmentation patterns of the glucocorticoid receptor from human brain and rat brain were very similar when analyzed by isoelectric focussing.The hormone specificity of the glucocorticoid receptor in human brain and in rat brain cytosol was compared by competition experiments using unlabelled dexamethasone, betamethasone, cortisol and corticosterone as competitors. No difference between human brain and rat brain cytosol was detected.It is concluded that the hormone specificity and the protein structure of the glucocorticoid receptors in human brain and in rat brain are similar.     

Rat-PET study without anesthesia: anesthetics modify the dopamine D1 receptor binding in rat brain

Positron emission tomography (PET) measurements in 6-month-old F344/N rats were performed in the conscious state and the influence of chloral hydrate, ketamine, and pentobarbital anesthesia on dopamine D(1) (DA-D(1)) receptor binding was evaluated using [(11)C]SCH23390, a selective DA-D(1) receptor ligand. To perform the PET study in conscious rats, an original fixation apparatus was developed and the animals were trained to acclimate to the scanning atmosphere for 3 h. This training was carried out twice a day for 2 weeks. PET measurements in conscious rats were successful, since the trained rats scarcely moved during the scanning (as monitored by video camera) and since highly reproducible measurements of binding potential (BP) were derived from their scanning. Chloral hydrate and ketamine anesthesia significantly increased the striatal BP of DA-D(1) receptors by 36% and 46%, respectively, compared to that observed in the conscious state. In contrast, pentobarbital markedly decreased the BP by 41%. These BP values of DA-D(1) receptors were calculated using a curve-fitting method. Our results indicate that PET studies in rats should be performed in the conscious state since the anesthetics dramatically modified ligand-receptor bindings, such as DA-D(1) receptor binding, in rat brain.     

Pharmacological characterization of serotonin receptor induced by rat brain messenger RNA in Xenopus oocytes

Serotonin receptors incorporated in the membrane of Xenopus oocytes injected with mRNA extracted from the rat brain was investigated b by intracellular recording. Serotonin elicited the membrane depolarization accompanied with membrane potential fluctuations. This serotonin action was suppressed by serotonin antagonists such as methysergide, cyproheptadine and ketanserin. Dibutyryl cyclic AMP and papaverine depolarized the membrane as seen in applying serotonin. These observations indicate that serotonin actions might involve the cAMP system.