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Our previous study showed an increase in total cholesterol level of the hippocampus after kainate-induced injury, but whether this is further metabolized to neurosteroids is not known. The first step in neurosteroid biosynthesis is the conversion of cholesterol to pregnenolone by the enzyme cytochrome P450 side chain cleavage (P450scc). This study was carried out to elucidate the expression of this enzyme in the kainate-lesioned rat hippocampus. A net decrease in P450scc protein was detected in hippocampal homogenates by Western blots at 2 weeks post-kainate injection (time of peak cholesterol concentration after kainate injury). Immunohistochemistry showed decreased labeling of the enzyme in neurons, but increased expression in a small number of astrocytes. The level of pregnenolone was also analyzed using a newly developed gas chromatography-mass spectrometry (GC-MS) method, optimized for the rat hippocampus. A non-significant tendency to a decrease in pregnenolone level was detected 2 weeks post-lesion. This is in contrast to a large increase in oxysterols in the lesioned hippocampus at this time (He et al. 2006). Together, they indicate that increased cholesterol in the kainate lesioned hippocampus is mostly metabolized to oxysterols, and not neurosteroids.

Peroxynitrite (ONOO(-)) and species derived from it can oxidize and nitrate lipids, proteins and DNA leading to changes in signaling molecules. The present study was carried out to elucidate possible effects of CNS peroxynitrite in a mouse model of orofacial pain. Mice that received facial carrageenan injection + intracerebroventricular (i.c.v.) injection of the peroxynitrite scavenger [5,10,15,20-Tetrakis (4-sulfonatophenyl) porphyrinato iron (III), chloride] (FeTPPS) showed significantly fewer face wash strokes upon probing the inflamed area of the face with a von Frey hair at 6 h after injection, compared to mice that received facial carrageenan alone, or facial carrageenan injection + i.c.v. injection of saline. Mice that received i.c.v. injection of FeTPPS without facial carrageenan injection showed no significant difference in response to von Frey hair stimulation, compared to mice that received i.c.v. injection of saline without facial carrageenan injection. These results indicate an anti-nociceptive effect of the peroxynitrite scavenger FeTPPS in carrageenan induced facial pain but no effect on normal tactile sensation and point to an important role of CNS peroxynitrite in nociception.

Increases in several ceramide species have been shown by non-targeted lipid profiling (lipidomics) of the rat hippocampus after kainate lesions (Guan et al. FASEB J 20:1152-1161, 2006). This study was carried out to examine possible effects of ceramide species on exocytosis. Significant increase in membrane capacitance in voltage-clamped rat pheochromocytoma (PC12) cells, an indication of exocytosis, was detected immediately after external application of C2, C6, and C18 ceramide. In contrast, no increase in capacitance was found after addition of C16 and C20 ceramide, or DMSO vehicle. The effect of ceramide on exocytosis was dependent on the integrity of lipid rafts. Treatment of cells with the cholesterol binding agent/disruptor of lipid rafts, methyl beta cyclodextrin, prior to addition of C18 ceramide suppressed the increase in capacitance induced by this lipid species. The ability of C2, C6 and C18 ceramide to trigger exocytosis was confirmed using total internal reflection fluorescence microscopy (TIRFM) experiments. External application of these species caused an exponential decrease in the number of subplasmalemmal neuropeptide Y (NPY)-enhanced green fluorescence protein (EGFP) labeled vesicles, indicating exocytosis. Interestingly, C18 is also the ceramide species that showed the greatest increase in the rat hippocampus after kainate excitotoxicity. It is postulated that C18 ceramide might facilitate exocytosis of glutamate from damaged neurons, thus propagating neuronal injury.

The present study was carried out to determine the effects of central nervous free fatty acids, lysophospholipids, or platelet activating factor (PAF), in a mouse facial carrageenan injection model of orofacial pain. Mice that received intracerebroventricular (I.C.V.) injection of arachidonic acid or oleic acid showed significantly reduced allodynia and behavioral responses to von Frey hair stimulation of a carrageenan-injected area of the face, at 8 h post-injection, compared to controls that received I.C.V. injection of vehicle. In contrast to free fatty acids, increased responses were observed in mice at 72 h after I.C.V. lysophosphatidic acid or lysophosphatidylcholine injection, and at 8 and 24 h after PAF injection, compared vehicle injected controls. Information regarding pro-nociceptive effect of specific brain lipids may be a useful basis for further studies to explore mechanism.

An increase in iron level, number of iron positive cells and ferritin expression has been observed in the rat hippocampus after neuronal injury induced by the excitotoxin, kainate. This is accompanied by an increased expression of divalent metal transporter-1 (DMT1) in the lesioned hippocampus, suggesting that the transporter may be partially responsible for the iron accumulation. DMT1 has a broad substrate range that includes other divalent metals such as lead (Pb) and cadmium (Cd), and the present study was carried out to elucidate the uptake of these metals in the kainate-injected brain. The technique of atomic absorption spectroscopy was used for analyses. Significantly higher lead and cadmium levels were detected in the hippocampus and other brain areas of intracerebroventricular kainate-injected rats treated with lead and cadmium in the drinking water, compared to intracerebroventricular saline-injected rats treated with lead and cadmium in the drinking water. Since very low levels of lead and cadmium are present in the normal animal, these results indicate increased uptake of lead and cadmium into brain areas as a result of the kainate injections. Increased iron levels were also detected in the hippocampus of the kainate-injected rats. The above results show increased uptake of divalent metals into brain areas undergoing neurodegeneration.

Iron regulatory proteins (IRP1 and IRP2) bind to iron response elements (IRE) on specific mRNAs, to affect the translation of many proteins involved in iron metabolism. An increase in iron levels and divalent metal transporter-1 (DMT1) expression have been observed in the rat hippocampus after excitotoxic injury induced by kainate, but thus far, it is not known whether these could be associated with changes in IRPs. The present study was therefore carried out to elucidate the expression of IRP1 or IRP2 and the IRE or non-IRE forms of DMT1 (DMT1 or -IRE DMT1) in the hippocampus after neuronal injury induced by kainate. A sustained upregulation of IRP1, IRP2, DMT1 and -IRE DMT1 protein was detected in the lesioned hippocampus by western blot and immunohistochemical analyses up to 2 months post-injection. Double immunofluorescence labeling showed that IRP1, IRP2, DMT1 and -IRE DMT1 were mostly expressed in GFAP positive astrocytes. The increased IRP expression could lead to increased expression of the +IRE form of DMT1. On the other hand, the increased expression of the -IRE DMT1 indicates that IRPs are unlikely to be only factor determining the expression of DMT1. It is postulated that transcription factors acting on putative AP-1, NF-kappaB binding sites, or gamma-interferon responsive elements on the DMT1 promoter may also play a role in upregulating the expression of the transporter. This could lead to increased iron influx into the brain areas undergoing neurodegeneration, and might be a factor contributing to neuronal damage after the initial excitotoxic injury.

Potentiation of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor function has been proposed as being useful in the treatment of depression, but thus far, little is known about the possible changes in AMPA receptor expression in the brain, after antidepressant treatment. The present study was carried out to study the expression of AMPA receptor subunits in different brain regions of mice that had been chronically injected with maprotiline. The latter is a modified tricyclic antidepressant that functions as a noradrenaline uptake inhibitor. Daily intraperitoneal injection with 10 mg/kg maprotiline for 30 days resulted in significantly increased GluR1 and GluR2/3 subunit expression in the nucleus accumbens and dorsal striatum as detected by immunohistochemistry; and significantly increased GluR1 and GluR2/3 expression in the hippocampus, as demonstrated by Western blot analysis. No change, or a decrease in GluR2 expression was detected in all the brain regions by both immunohistochemistry and Western blots. The increase in GluR1 and GluR2/3, but no increase in GluR2 subunits suggests that there could be an increase in calcium permeability of AMPA receptors in limbic/striatal brain regions after maprotiline treatment. This could lead to increased synaptic activity or plasticity in the hippocampus and striatum, and may underlie the therapeutic effect of maprotline, and possibly, other antidepressant drugs.

A gradual increase in iron occurs in the lesioned hippocampus after neuronal injury induced by the excitotoxin kainate, and the present study was carried out to investigate whether this increase in iron might be associated with changes in expression of the iron binding protein, ferritin. An increase in ferritin immunoreactivity was observed in glial cells of the hippocampus, as early as three days after intracerebroventricular injections of kainate. The number of ferritin positive cells peaked four weeks after the kainate injection, and decreased eight and twelve weeks after injection. They were found to be mostly microglia and oligodendrocytes by double immunofluorescence labeling with glial markers. A number of ferritin-labeled endothelial cells were also observed via electron microscopy. The decline in ferritin immunoreactivity four weeks after the injection of kainate is accompanied by an increase in the number of ferric and ferrous iron positive cells in the lesioned tissue. A substantial non-overlap between ferritin and iron-containing cells was observed. In particular, spherical ferric or ferrous iron-laden cells in the degenerating hippocampus were unlabeled for ferritin for long time periods after the kainate injection. An increase in iron, together with a reduced expression of iron binding proteins such as ferritin at long time intervals after kainate lesions, could result in a relative decrease in ferritin-induced ferroxidase activity and the presence of some of the iron in the ferrous form. It is postulated that this may contribute to chronic neuronal injury, following acute kainate-induced neurodegeneration.

The present study aimed to elucidate sPLA(2) activity in the normal and kainate-lesioned hippocampus using selective inhibitors of sPLA(2). In normal rats the highest levels of sPLA(2) were observed in the hippocampus, pons, and medulla, followed by the cerebral neocortex and caudate nucleus. After intracerebroventricular kainate injections an increase in total PLA(2) activity was observed in the rat hippocampus. Using a selective sPLA(2) inhibitor 12-epi-scalaradial, sPLA(2) activity was found to be significantly increased by 2.5-fold on the side of the intracerebroventricular injection compared to the contralateral side. A peptide P-NT.II, derived from the amino acid sequence of "PLA(2)-inhibitory protein," discovered in the serum of the reticulated python, also showed potent sPLA(2) inhibitory activity in homogenates from the kainate-injected hippocampus. These results show that there is a high level of sPLA(2) activity in the normal hippocampus, pons, and medulla oblongata, and that the level increases further in the hippocampus after kainate-induced excitotoxic injury. The increased PLA(2) activity was inhibited by P-NT.II, indicating a potential use of this peptide as a PLA(2) inhibitory agent in the brain.

The present investigation was carried out to study the possible effects of quinacrine in modulating cytoplasmic phospholipase A(2) (cPLA(2)) mRNA levels in rat hippocampus after kainate treatment. Injections of kainate into the right lateral ventricle resulted in significant increases in cPLA(2) mRNA levels in the hippocampus, at 3 days and 7 days after injection. The elevation in cPLA(2) mRNA levels is consistent with previous observations of increased cPLA(2) immunoreactivity in degenerating neurons and astrocytes at these times. Rats that received once daily intraperitoneal injections of quinacrine (5 mg/kg) after the intracerebroventricular kainate injections showed almost complete attenuation of increased cPLA(2) expression, at both 3 and 7 days after kainate injection. These results show that in addition to its well-known effect of inhibition of PLA(2) activity, quinacrine could also inhibit cPLA(2) expression, and further supports a role for PLA(2) in kainate-induced neuronal injury.

The present study aimed to elucidate the subcellular distribution of the Cdc42 and Rac activated alpha and beta isoforms of p21-activated kinase (PAK) in the monkey cerebral neocortex and hippocampus. These proteins have been shown to play morphological roles through effects on the actin cytoskeleton. alphaPAK immunoreaction product was concentrated in regions of axon terminals or dendrites, some distance (0.2-1 microm) away from the synapse. The labeled portion of dendrite often appeared with "ruffled" cell membranes or resembling non-synapse forming "buds." betaPAK immunoreaction product was concentrated in cell bodies and larger diameter dendrites. Immunogold labeled sections showed that most of the label for both alphaPAK and betaPAK was present in a perisynaptic or extrasynaptic location, and relatively little staining was present on the postsynaptic density. Because alphaPAK has been shown to be associated with new membrane structures involving the Rho family GTPase Rac1, which controls dendritic morphology, these observations suggest alphaPAK positive regions of axons and dendrites may mark new areas of neurite extension.

The present study aimed to elucidate the distribution of ferric and ferrous iron in the hippocampus after kainate-induced neuronal injury. A modified Perl's or Turnbull's blue histochemical stain was used to demonstrate Fe3+ and Fe2+ respectively. Very light staining for iron was observed in the hippocampus, in normal or saline-injected rats and 1-day post-kainate-injected rats. At 1 week postinjection, a number of Fe3+-positive, but very few Fe2+-positive, cells were present, in the degenerating CA fields. At 1 month postinjection, large numbers of Fe3+-positive glial cells, and some Fe2+-positive blood vessels, were observed. At 2 months postinjection, large numbers of Fe3+- and Fe2+-positive glial cells were present. The labeled cells had light and electron microscopic features of oligodendrocytes, and were double labeled with CNPase, a marker for oligodendrocytes. The observation of an increasing number of Fe3+- and Fe2+-positive cells in the degenerating hippocampus with time is consistent with the results of a nuclear microscopic study, in which an increasing amount of iron was detected in the degenerating hippocampus after kainate injection. In addition, the present study showed a shift in the oxidation state of the accumulated iron, with more cells becoming Fe2+ at a late stage. A possible consequence of the high amounts of Fe2+ in the hippocampus after kainate injection is that it could promote free radical damage in the lesioned areas.

Niemann-Pick disease type C (NP-C) is an inherited neurodegenerative disorder associated with intracellular cholesterol and glycolipid trafficking defects. Two separate genes, NPC1 and NPC2, have been linked to NP-C. NPC1 encodes a polytopic membrane-bound protein with a putative sterol-sensing domain. NPC2 has been recently identified as epididymal secretory glycoprotein 1. The NPC1 protein functions in the vesicular redistribution of endocytosed lysosomal cargo, but how its inactivation leads to neurodegeneration is not known. The neurological symptoms of NP-C typically appear after a period of normal early development and reflect progressive degeneration of widespread brain regions. Here we have delineated the pattern of neurodegeneration in NP-C mice, whose genetic defect has been shown to be an inactivating mutation of the mouse NPC1 gene. The results reveal a spatially and temporally specific pattern of degeneration of nerve fibers followed by degeneration of neuronal cell bodies beginning as early as day 9 and continuing throughout life. We have recently showed that in the primate brain, the NPC1 protein is localized predominantly within perisynaptic astrocytic processes. The present observations suggest that a functional disturbance in NPC1 could disrupt vesicular transport of cholesterol, glycolipids and possibly other endocytic cargo in glia, which is critical for maintaining the integrity of neurons.

The distribution of cytoplasmic phospholipase A2 (cPLA2), 4-hydroxynonenal (HNE), and choline acetyltransferase (ChAT) was studied in the septum and hippocampus of rats at various time intervals after fimbria-fornix (FF) transection. Very little cPLA2 or HNE immunoreactivity was observed in the normal medial or lateral septum, whereas a large increase in immunoreactivity with both antibodies was observed in the lateral septum one week after transection. The increase in cPLA2 or HNE staining in the lateral septum after FF transection was completely blocked by intraperitoneal injections (once daily) of a lipophilic inhibitor of phospholipase A2, quinacrine (5 mg/kg), showing the importance of phospholipase A2 in generation of arachidonic acid, which is a target for lipid peroxidation and formation of 4-hydroxynonenal. Quinacrine prevented not only a rise in HNE immunoreactivity, but also a rise in cPLA2 immunoreactivity, showing that cPLA2 expression itself is depressed by the drug, in addition to its well-known effect on blocking the catalytic action of phospholipase A2. No increase in cPLA2 or HNE immunoreactivity was observed in neurons of the medial septum after fimbria-fornix transection, even though these showed a decrease in ChAT staining after the lesion. This suggests that glutamate released from transected hippocamposeptal afferents or increased activity of the supramammillary area following FF transection may lead to increased cPLA2 and HNE immunreactivity, whereas retrograde degeneration in neurons may not. We conclude that there is free-radical damage, as evidenced by HNE formation in neurons of the lateral septum after fimbria-fornix transection, and that this increase in HNE is dependent on phospholipase A2 activity.

The present study aimed to elucidate the distribution of heme oxygenase-1 (HO-1) in the hippocampus after intracerebroventricular injections of kainate. Very little or no staining of HO-1 was observed in the normal CA1, whilst moderate staining of dentate hilar neurons was observed in the dentate gyrus, in the normal hippocampus. At postinjection day 1, a slight increase in immunoreactivity in the neuropil of the lesioned CA fields and a marked increase in HO-1 immunoreactivity in glial cells of the stratum lacunosum moleculare of CA fields and the stratum moleculare of the dentate gyrus was observed. Electron microscopy showed that the glial cells had features of viable astrocytes. At postinjection day 3, glial cells in the dentate gyrus continued to express HO-1, whilst pyramidal neurons in the degenerating CA fields started to express intense HO-1 immunoreactivity in their cell bodies. At postinjection weeks 1-3, HO-1 was observed in glial cells in the center of the lesion, but also in neurons at the perifocal region of the glial scar. The glial cells were found to have features of viable astrocytes and microglia, whilst the neurons contained discontinuous cell membranes and nuclear outlines, and had features of degenerating neurons. Intense immunoreactivity was observed in the cytoplasm of the degenerating neurons. The density of staining was greater than that observed in astrocytes or microglia. Recent in vitro results on fibroblasts transfected with HO-1 cDNA showed that, despite cytoprotection with low (less than fivefold compared with untransfected cells) HO-1 activity, high levels of HO-1 expression (more than 15-fold) were associated with significant oxygen toxicity. These and the present observations suggest a destructive effect of increased expression of HO-1 in neurons, and possible novel therapeutic approaches involving overexpression of HO-1 must therefore be approached with caution.

Lipid peroxidation is known to be associated with many neurodegenerative diseases and with traumatic brain injury, but its occurrence in the normal developing brain has not been reported. The present study was carried out using a specific antibody that recognises proteins modified by the end-product of lipid peroxide decomposition, 4-hydroxynonenal (HNE), to evaluate evaluate possible lipid peroxidation products in the brains of developing rats by immunocytochemistry and electron microscopy. Moderately dense labelling was observed in the supraventricular corpus callosum in the 7- and 8-day-old rats, whilst very dense labelling was observed in the same region, in the 9- and 10-day-old rats. Very little immunoreactivity was observed at 14 days, and no staining was observed in the corpus callosum in adult rats. HNE staining was not observed in neuronal cell bodies that give rise to callosal axons in the overlying cerebral cortex. Electron microscopy showed dense HNE staining on the basal laminae of blood vessels and on the plasma membranes of unmyelinated axons. Large numbers of rounded cells with features of oligodendrocyte precursor cells were labelled by Perl's stain in the supraventricular corpus callosum at postnatal day 7 and postnatal day 10, i.e. at times corresponding to high levels of HNE immunoreactivity. In contrast, very few such cells were observed in the adult brain, corresponding to the very little or no Perl's staining in the adult. These results suggest that lipid peroxidation observed in the supraventricular corpus callosum at postnatal day 10 could result from an accumulation of iron in this region, at this time.

The aim of the present study was to elucidate the distribution of glutathione immunoreactivity in the normal hippocampus and after kainate-induced neuronal injury. A specific antibody was used that recognizes both the reduced (GSH) and oxidized (GSSG) forms of glutathione. Immunoreactivity to glutathione was observed in neurons, but few immunolabeled glial cells were observed in the normal hippocampus. After kainate injection, a decrease in glutathione immunoreactivity was observed in pyramidal neurons from as early as 1 day after injection. In contrast, dense staining to glutathione was observed in large numbers of reactive astrocytes at 3 days to 6 weeks after kainate injection. This suggests upregulation of glutathione synthesis in these cells. One possibility is that the high content of glutathione is protective to reactive astrocytes. Another possibility is that the high glutathione concentration in reactive astrocytes may be protective to neurons around the glial scar.

Intracerebroventricular kainate treatment in rats induces neuronal cell death, followed by proliferation and hypertrophy of glial cells in the lesioned area. To further understand the activated signal transduction pathways and to get insights into potential target gene activation, the present study aims to elucidate long-term effects on the phosphorylation state of cAMP response element-binding protein (CREB) in the hippocampal formation. One to four weeks after kainate injection, we found high levels of phosphorylated and hence activated CREB (pCREB) in glial cells of the degenerating CA fields. As shown by electron microscopy, pCREB immunoreactivity was present in reactive astrocytes, oligodendrocyte precursor cells and endothelial cells of blood vessels. It is postulated that pCREB could drive the expression of downstream genes in these cells to promote cell proliferation and survival.

The expression of P-glycoprotein (PGP) was studied by immunocytochemistry and light and electron microscopy, in normal rats and after intracerebroventricular kainate injections. Two antibodies to PGP, mdr (Ab-1) and c-219, were used. As in previous studies (Thiebault et al. and Jette et al.), labelled capillaries were observed in normal rats. Kainate injections resulted in death of pyramidal neurons in the hippocampus, and a proliferation of glial cells in the affected cornu ammonis fields. An increase in PGP expression was observed in reactive astrocytes as early as 1 day postinjection. Immunoreactivity peaked at 2 weeks postinjection, but was still visible as late as 10 weeks postinjection. Similar results were observed using the two antibodies. Double immunolabelling and confocal microscopy also showed that PGP was colocalised with GFAP, a marker for astrocytes. The expression of PGP in astrocytes was confirmed by electron microscopy, which showed immunoreaction product in cells containing dense bundles of glial filaments and features of reactive astrocytes. The increased PGP expression in reactive astrocytes could be part of a cellular stress response program in these cells.

Specimens of human cerebral cortex were obtained during neurosurgical operations and studied by immunocytochemistry and electron microscopy, using antibodies to the metabotropic glutamate receptor subunit mGluR1a and the ionotropic glutamate receptor GluR2/3. A small number of non-pyramidal neuronal cell bodies were labelled for mGluR1a. Double immunolabelling with mGluR1a and GluR2/3 showed that most pyramidal cell bodies were labelled for GluR2/3 but not for mGluR1a. Despite the non-colocalisation of these two receptor subtypes in cell bodies, however, many dendrites and dendritic spines were double-labelled for mGluR1a and GluR2/3 at electron microscopy. As there is evidence that most neurons positive for GluR2/3 are pyramidal cells, this suggests that mGluR1a is present in dendrites of pyramidal neurons, despite absent or low levels of immunoreactivity in their cell bodies.

The distributions of the kainate/DL-alpha-amino-3-hydroxy-5-methylisoxazolepropionic acid (KA/ AMPA) receptors GluR1 and calcium-activated neutral protease II (calpain II) in the hippocampus of normal and kainate-lesioned rats were studied by immunocytochemistry. There was a reduction in GluR1 immunoreactivity and a slight increase in calpain II immunoreactivity on the dendrites of pyramidal neurons in CA fields affected by the kainate at 18 h postinjection. Calpain II immunore-activity was associated with amyloid fibrils at electron microscopy. These fibrils were most often intracellular, in membrane-bound profiles, some of which were contacted by axon terminals and were identified as degenerating dendrites. There was extensive destruction of mitochondrial membranes in degenerating profiles, and accumulations of amyloid fibrils were often localised in mitochondria in a calpain-positive profile. This was unlike other, calpain-negative degenerating profiles, that contained tubulovesicular profiles or multilamellar bodies, where mitochondrial membranes were preserved. Many more calpain-positive profiles were observed at electron microscopy 6 days after kainate injection. The enzyme was present in macrophages and astrocytes in lesioned areas.

Specimens of human cerebral neocortex were obtained during neurosurgical operations and studied by immunocytochemistry and electron microscopy, using antibodies to the glutamate receptor subunit GluR1 and gamma-aminobutyric acid (GABA). Many GluR1-positive pyramidal neurons and fewer GluR1-positive non-pyramidal neurons were present in the cortex. Non-pyramidal neurons were more heavily labelled for GluR1 than pyramidal neurons. Most GABAergic neurons were labelled for GluR1. The white matter was unstained, except for occasional labelled neurons. This pattern of GluR1 immunostaining is similar to that in rat cerebral cortex, but is different from that in the hippocampus and amygdala, where large numbers of pyramidal or projection neurons, but few non-pyramidal or GABAergic neurons, were labelled for GluR1.

The distribution of the [3H]alpha-amino-3-hydroxy-5-methylisoxzalepropionic acid (AMPA) receptor subunit GluR4 was studied in frontal, parietal and temporal cerebral cortex, subcortical white matter and corpus callosum of neonatal, immature and mature rats. In 1- to 2-day-old rats, a few oligodendrocyte progenitors and amoeboid microglia in the supraventricular part of the corpus callosum were immunolabelled for GluR4. At 7 to 10 days, the number of amoeboid microglia and oligodendrocyte progenitors in white matter increased; many neurons in cortex, including pyramidal neurons, were also moderately labelled for GluR4. The pattern of GluR4 immunostaining in 14-day-old rats was different from that in 7- to 10-day-old rats, but similar to the adult, in that there was no immunoreactivity in microglia and oligodendrocyte progenitors in subcortical white matter. A proportion of non-pyramidal neurons in cortex were moderately labelled, while some pyramidal neurons were lightly labelled. A population of small glial cells with features of oligodendrocyte progenitors were densely labelled in cortex.

Immunocytochemistry was used to study the distribution of the kainate receptors GluR1, GluR2/3 and GluR4 and of the N-methyl-D-aspartate (NMDA) receptor NMDAR1 as well as the astrocyte markers glutamine synthetase (GS) and glial fibrillary acidic protein (GFAP) in the hippocampus of normal and kainate-lesioned rats. Hippocampal pyramidal neurons and dentate granule neurons were labelled heavily for GluR1 and GluR2/3, but only lightly for GluR4. Dense GluR4 immunopositivity was, however, observed in oligodendrocyte-like glial cells. Hippocampal pyramidal neurons and dentate granule neurons were moderately labelled for NMDAR1. Intravenous kainate injections resulted in a decrease in GluR1 and GluR2/3 immunoreactivity on the apical dendrites of pyramidal neurons as early as 7 h postinjection. At 18 h, there was a marked reduction in GluR1 and GluR2/3 receptors in the terminal tuft of dendrites of most hippocampal pyramidal neurons in the affected area, although some cells showed labelling in other portions of the apical dendrites and in basal dendrites. Immunostaining for GluR4 and NMDAR1 was also reduced at this time. At postinjection day 3, only the cell bodies and the basal dendrites of a few scattered pyramidal cells were labelled. Taken together, these results indicate a progressive loss of glutamate receptors, which affects the apical dendritic tree before the basal dendritic tree. The decrease in receptor immunoreactivity could be due to a downregulation of the receptors, since it occurred as early as 7 h postlesion, before cell death was evident in Nissl-stained sections. At long intervals after kainate injection, all pyramidal cells at the centre of the lesion showed a lack of glutamate receptor staining, and no partially labelled pyramidal cells were observed. The periphery of the lesion, however, contained many partially labelled pyramidal neurons among the unlabelled cells and had features of early lesions. The present study also showed an early decrease in GS immunoreactivity in the affected CA fields of the hippocampus (18 h to 3 days postinjection), followed by a medium-term increase (5-68 days) and a late decrease in GS immunoreactivity (81 days). The decrease in GS immunoreactivity at 81 days is not due to an absence of astrocytes, since GFAP staining showed many densely labelled astrocytes in the affected CA field.