Alteration in the immunoreactivity of the calcineurin subunits after ischemic hippocampal damage.

Dephosphorylation processes of target proteins are critical to the reversible regulation of intracellular signal transduction systems. Further, brain damage such as ischemic insult induces marked changes in protein kinase activity. To study these changes more thoroughly, specific monoclonal antibodies of the A and B subunits of calcineurin (protein phosphatase 2B) were raised, and regional alterations in the immunoreactivity of calcineurin in the rat hippocampus were investigated after a transient forebrain ischemic insult causing selective and delayed hippocampal CA1 pyramidal cell damage. In normal rats it was found that both the calcineurin A and the B subunits showed high immunoreactivity in the dendritic fields of the hippocampal formation. The immunoreactivity of subunit A in the strata oriens, the radiatum of the CA1 subfield and in the stratum lucidum of the CA3 subfield was most intense, whereas the immunoreactivity in the other CA3 subfields and in the dentate gyrus was relatively low. In contrast, the dendritic fields of the hippocampal formation were equally immunoreactive to calcineurin subunit B, although the stratum lucidum of the CA3, where the mossy fibers from the dentate granule cells terminate, showed a very high immunoreactivity of the B subunit. After transient forebrain ischemia in the CA1 subfield, where selective pyramidal cell death occurred two days after this ischemia, a marked loss of immunoreactivity in both subunits was observed, along with morphological pyramidal cell damage. A recovery of the immunoreactivity of A and B subunits in the strata oriens and radiatum was later noted 30 days after ischemia. In the stratum lucidum of the CA3, the immunoreactivity of both the A and B subunits was transiently depressed from 6 to 24 h, followed by a marked immunoreactivity enhancement from four to 30 days after ischemia. Further, in the histologically intact dentate gyrus, both the immunoreactivity of the A and B subunits in the molecular layer were transiently enhanced from four to 14 days after ischemia, particularly in the supragranular layer. The results clearly indicate that the protein dephosphorylation systems were markedly altered in the whole hippocampal formation during the recirculation period following ischemia. Further, the transient depression in the calcineurin immunoreactivity seen in the mossy fiber terminals may reflect modulated synaptic activity of the dentate granule cells, which may play a pivotal role in the delayed and selective death of the CA1 pyramidal cells. Thus, calcineurin appears to be an excellent marker enzyme for the detection of neuronal activity and synaptic plasticity after brain damage, such as an ischemic insult.     

Protein disulfide isomerase immunoreactivity and protein level changes in neurons and astrocytes in the gerbil hippocampal CA1 region following transient ischemia

We investigated the temporal and spatial alterations of protein disulfide isomerase (PDI) immunoreactivity and protein level in the hippocampus proper after 5 min transient forebrain ischemia in gerbils. PDI immunoreactivity was significantly altered in the hippocampal CA1 region. PDI immunoreactivity in the sham-operated animals was found in non-pyramidal cells. At 30 min after ischemia, PDI immunoreactivity was shown in the pyramidal cells of the stratum pyramidale (SP): the PDI immunoreactivity in the pyramidal cells was increased up to 12 h after ischemia. Thereafter PDI immunoreactivity was decreased, and the PDI immunoreactivity was shown in non-pyramidal cells 2 days after ischemia. Four to 5 days after ischemia, almost pyramidal cells in the CA1 region were lost because the delayed neuronal death occurred. At this time period, PDI immunoreactivity was expressed in some astrocytes as well as some neurons. The results of the Western blot analysis were consistent with the immunohistochemical data. These findings suggest that increase of PDI in pyramidal cells may play a critical role in resistance to ischemic damage at early time after ischemic insult, and that expression of this protein in astrocytes at late time after ischemic insult is partly implicated in the acquisition of tolerance against ischemic stress.     

Structural characteristics of astrocytes from the rat hippocampus in postischemia

The aim of this investigation was to study the distribution and structural organization of rat hippocampal astrocytes containing immunoreactive glial fibrillary acidic protein (GFAP) after ischemic damage of the brain in the animals treated with intraventricular infusion of creatine as a neuroprotective drug, and in those which received no treatment. Using the methods of light microscopy and immunocytochemistry, the brain of 26 mature Sprague-Dawley (Koltushi) rats was studied. Some animals were narcotized and subjected to general brain ischemia (lasting for 12 min) followed by a reperfusion (for 7 days). Creatine was infused intraventricularly to 11 animals using an automatic Alzet osmotic minipump. It was found that GFAP-immunoreactive hippocampal astrocytes were concentrated within two major areas (stratum lacunosum-moleculare CA1 and fascia dentata stratum polymorphae). As a result of neuroprotective effect of creatine, moderate ischemic damage of the hippocampus was not followed by the changes in the zones of activated astrocyte localization. Redistribution of GFAP-positive astrocytes in postischemic period was caused by the loss of pyramidal neurons in cytoarchitectonic field CA1. Complete loss of pyramidal neurons in this hippocampal area resulted in a qualitatively new level of astrocyte activation--their proliferation.     

Glutamate decarboxylase-immunoreactive neurons in the aging rat hippocampus are more resistant to ischemia than CA1 pyramidal cells

Glutamate decarboxylase (GAD)-immunoreactive, supposedly GABAergic inhibitory, neurons in various fields of the rat hippocampus and pyramidal cells in area CA1 were quantified 1 week after transient cerebral ischemia by 4-vessel occlusion. Whereas the number of CA1 pyramidal cells in Toluidine blue-stained semithin sections were found reduced by 50% when compared with controls there was no loss of GAD-immunoreactive cells in vibratome sections of hippocampus proper and fascia dentata. These data suggest that GABAergic hippocampal neurons are more resistant to ischemia than CA1 pyramidal cells.     

Calbindin-D28k在出生前人海马本部及下托神经元的表达及其变化

本实验采用免疫组织化学方法研究了１３～３８ 周人胎儿海马本部及下托含Ｃａｌｂｉｎｄｉｎ－Ｄ２８ｋ 神经元的分布和发育。结果表明：在１３～１４ 周时,许多含Ｃａｌｂｉｎｄｉｎ－Ｄ２８ｋ 锥体细胞可见于ＣＡ１ 区锥体细胞层中部及深部,随着胎龄增大,ＣＡ１ 区含Ｃａｌ－ｂｉｎｄｉｎ－Ｄ２８ｋ 锥体细胞的数量及密度逐渐下降,最终消失,并且这种下降及消失首先从含Ｃａｌｂｉｎｄｉｎ－Ｄ２８ｋ 锥体细胞区浅部开始,然后向深部推进;在１３～２８ 周期间,ＣＡ２ 和ＣＡ３ 区也有许多含Ｃａｌｂｉｎｄｉｎ－Ｄ２８ｋ 锥体细胞,但至３２ 周以及其后,ＣＡ３ 和ＣＡ２ 区则不见含Ｃａｌｂｉｎｄｉｎ－Ｄ２８ｋ 锥体细胞,仅在ＣＡ２ 与ＣＡ１ 交界区见到少量弱染的含Ｃａｌｂｉｎｄｉｎ－Ｄ２８ｋ 锥体细胞。此外,在２８～３８ 周期间,ＣＡ３ 和ＣＡ２ 区锥体细胞层周围可见许多含Ｃａｌｂｉｎｄｉｎ－Ｄ２８ｋ 的苔藓纤维,其密度随胎龄增大而增加。１４～３８ 周期间,许多含Ｃａｌｂｉｎｄｉｎ－Ｄ２８ｋ 的锥体细胞也出现于下托锥体细胞层全层及前下托锥体细胞层浅部（细胞岛区）及中部。这些区域含Ｃａｌ－ｂｉｎｄｉｎ－Ｄ２８ｋ 锥体细胞的数量及染色强度在１４～２４ 周期间逐渐增     

Structural organization of astrocytes in the rat hippocampus in the post-ischemic period

The aim of the present work was to study the location and structural organization of astrocytes in the rat hippocampus, which contain immunoreactive glial fibrillary acid protein (GFAP) after ischemic damage to the brain after intracerebroventricular administration of the neuroprotective agent creatine and without treatment. Light microscopy and immunocytochemical methods were used to study the brains of 26 adult male Sprague-Dawley (Koltushi) rats, some of which were subjected to total cerebral ischemia (12 min) under anesthesia with subsequent reperfusion (seven days). Creatine was given to 11 animals intracerebroventricularly using an osmotic pump (Alzet Osmotic Mini-Pump). The results showed that GFAP-immunoreactive hippocampal astrocytes were concentrated in two main zones (the stratum lacunosummoleculare of field CA1 and the stratum polymorphae of the dentate fascia). The neuroprotective effect of creatine had the result that moderate ischemic damage to the hippocampus did not lead to changes in the zones containing activated astrocytes. The redistribution of GFAP-positive astrocytes in the post-ischemic period was associated with loss of pyramidal neurons in cytoarchitectonic field CA1. Complete loss of pyramidal neurons in this area of the hippocampus leads to a qualitatively new level of astrocyte activation - proliferation.Translated from Morfologiya, Vol. 125, No. 2, pp. 19–21, March–April, 2004.     

Changes of pyridoxal kinase expression and activity in the gerbil hippocampus following transient forebrain ischemia

In the previous study, we observed chronological alterations of glutamic acid decarboxylase (GAD), which is the enzyme converting glutamate into GABA. GAD isoforms (GAD65 and GAD67) differ substantially in their interactions with cofactor pyridoxal 5'-phosphate, which is catalyzed by pyridoxal kinase (PLK). In the present study, we examined the chronological changes of PLK expression and activity in the hippocampus after 5 min transient forebrain ischemia in gerbils. PLK immunoreactivity in the sham-operated group was detected weakly in the hippocampus. Ischemia-related change of PLK immunoreactivity in the hippocampus was significant in the hippocampal cornu ammonis (CA1)region, not in the hippocampal CA2/3 region and dentate gyrus. PLK immunoreactivity was observed in non-pyramidal GABAergic neurons at 30 min to 3 h after ischemic insult. At 12 h after ischemic insult, PLK immunoreactivity was shown in many CA1 pyramidal cells as well as some non-pyramidal cells. At this time point, PLK immunoreactivity and protein content was highest after ischemia. Thereafter, PLK immunoreactivity and protein content is decreased time-dependently by 4 days after ischemic insult. Four days after ischemia, some astrocytes expressed PLK in the CA1 region. The specific PLK activity was not altered following ischemic insult up to 2 days after ischemic insult. Thereafter, the specific PLK activity decreased time-dependently. However, total activity of PLK was significantly increased 12-24 h after ischemic insult, and thereafter total activity of PLK decreased. Therefore, we suggest that the over-expression of PLK in the CA1 pyramidal cells at 12 h after ischemia may induce increase of GAD in the CA1 pyramidal cells, which plays an important role in delayed neuronal death via the increase of GABA or enhancement of GABA shunt pathway.     

Inhibition of the canonical Wnt pathway by Dickkopf-1 contributes to the neurodegeneration in 6-OHDA-lesioned rats

We investigated the cellular localization and progressive changes of corticotropin releasing factor (CRF) in the mouse hippocampus, during and after pilocarpine induced status epilepticus (PISE) and subsequent epileptogenesis. We found that CRF gene expression was up-regulated significantly at 2h during and 1d after PISE in comparison to control mice. Immunohistochemical analysis showed that the number of CRF and Fos immunoreactive cells was increased significantly in the strata oriens and pyramidale of CA1 area and in the stratum pyramidale of CA3 area at 2h during and 1d after PISE. CRF was induced in calbindin (CB) or calretinin (CR) immunoreactive interneurons in stratum oriens at 2h during PISE. It suggests that induced CRF may be related to the over excitation of hippocampal neurons and occurrence of status epilepticus. It may also cause excitoneurotoxicity and delayed loss of CA3 and CA1 pyramidal neurons, leading to the onset of epilepsy.     

Long-term lack of endogenous glucocorticoids down-regulates glucocorticoid receptor levels in the rat forebrain

To understand the effect of a chronic lack of endogenous glucocorticoids on glucocorticoid receptor levels, the changes of glucocorticoid receptor content in the rat forebrain five months after adrenalectomy were investigated. In the long-term adrenalectomized rats that showed a hormone deficiency and loss of glucocorticoid receptor immunoreactivity in the forebrain, an intraperitoneal injection of corticosterone was used to elevate the serum hormone levels and recover glucocorticoid receptor immunoreactivity in the forebrain. One hour later, when the blood corticosterone returned to the normal level, the recovery of glucocorticoid receptor immunoreactivity in the forebrain was examined by immunohistochemistry. Since the complete restoration of glucocorticoid receptor immunoreactivity was shown to depend on the presence of normal levels of both serum hormone and intracellular glucocorticoid receptors, the weak reappearance of glucocorticoid receptor immunoreactivity in any forebrain area of the long-term adrenalectomized rats that had normal serum corticosterone might reflect the low intracellular glucocorticoid receptor levels there. Our results revealed a weak reappearance of glucocorticoid receptor immunoreactivity in some forebrain areas of the long-term adrenalectomized rats after corticosterone treatment; the hippocampal granule cell layer and cerebral cortex in particular showed very weak recovery of glucocorticoid receptor immunoreactivity. Conversely, neurons in the CA1/CA2 subfields of the hippocampal pyramidal cell layer, immediately adjacent to the granule cell layer on the same brain section, exhibited a strong reappearance of glucocorticoid receptor immunoreactivity, to near normal levels.

These results suggest that, five months after adrenalectomy, the intracellular glucocorticoid receptor content decreased in the rat granule cell layer and cerebral cortex. Therefore, the long-term lack of endogenous glucocorticoids after adrenalectomy might down-regulate but not up-regulate the intracellular glucocorticoid receptor level, and the presence of glucocorticoids is important for the continued synthesis of glucocorticoid receptors.     

Post-ischemic administration of progesterone in rats exerts neuroprotective effects on the hippocampus

Progesterone is neuroprotective in models of focal or global ischemia when treatment starts either before the insult or at the onset of reperfusion. In these cases the steroid may act during the occurrence of the early pathophysiological events triggered by ischemia or reperfusion. As opposed to this condition, the aim of the present study was to assess the effect of delayed, post-injury administration of progesterone on the preservation of pyramidal neurons of the hippocampus of rats 21 days after been exposed to global ischemia by the four vessel occlusion model. Progesterone (8 mg/kg, i.v.) or its vehicle, were administered at 20 min, 2, 6, and 24h after the end of ischemia. At histological examination, brains of the ischemic vehicle-treated rats showed a severe reduction of the population of pyramidal neurons in the CA1 and CA2 subfields (12% and 29% remaining neurons, respectively), and a less severe neuronal loss in the CA3 and CA4 subfields of the hippocampus (68% and 63% remaining neurons, respectively), as compared to rats exposed to sham procedures. They also showed a two-fold enlargement of the lateral ventricles and 33% shrinkage of the cerebral cortex as compared to the sham group. Progesterone treatment resulted in a significant preservation of pyramidal neurons in CA1 and CA2 (40% and 62% remaining neurons), with no ventricular dilation and only a mild (12%) cortical shrinkage. Results suggest that progesterone is able to interfere with some late pathophysiological mechanisms leading both to selective neuronal damage in the hippocampal CA1 and CA2 subfields, and to shrinkage of the cerebral cortex.     

Age-related alterations of GABAergic input to CA1 pyramidal neurons and its control by nicotinic acetylcholine receptors in rat hippocampus

The aim of this study was to determine whether age-associated alterations in the GABAergic input to pyramidal neurons in the hippocampus are due to a dysfunction of GABAergic interneurons, and/or a decrease in their cholinergic control via nicotinic receptors (nAChRs). Electrophysiological recordings were obtained from pyramidal cells in the CA1 area of hippocampal slices from young (3-4 months old) and aged (25-30 months old) Sprague-Dawley rats. Synaptic GABA(A) receptor-mediated inhibitory postsynaptic currents and inhibitory postsynaptic potentials induced by stimulation of the stratum oriens were significantly smaller in aged rats. The frequency (but not amplitude) of spontaneous and miniature GABA inhibitory postsynaptic currents (IPSCs) was reduced in aged rats, suggesting a presynaptic alteration. Tetanic stimulation of cholinergic afferents to release endogenous acetylcholine, or an exogenous application of the nAChR agonist cytisine, increased the frequency of spontaneous IPSCs in young rats; however these effects were not evident in aged rats, indicating that the nicotinic control of GABA release is lowered during aging. None of these age-related alterations were reversed by a chronic treatment with donepezil, a cholinesterase inhibitor. Immunofluorescent labeling of GABA interneurons with somatostatin (SOM), parvalbumin (PV) or calbindin (CB), together with the vesicular acetylcholine transporter VAChT, revealed a selective loss of subpopulations of SOM and CB positive interneurons. This loss was associated with a general decrease in density of the cholinergic network in aged rats. Thus, the lower GABAergic inhibition observed in the aged rat hippocampus is due to a selective loss/dysfunction of subpopulations of GABAergic interneurons, associated with a widespread cholinergic deficit.     

Expression of estrogen receptor-beta in the postischemic monkey hippocampus

The molecular basis of estrogen-mediated neuroprotection against brain ischemia remains obscure. Here, we studied by immunohistochemistry the expression of estrogen receptor (ER) alpha and beta in the hippocampal CA1 sector of postischemic adult macaque monkeys. ERbeta was present in control CA1 pyramidal neurons, decreasing on day 4 after ischemia. In contrast, ERbeta immunoreactivity increased remarkably in the radiate and molecular layers of CA1, where it was present in astrocytes and microglia. ERalpha was negligible in both control and postischemic monkeys. These results indicate that ERbeta is the major receptor responsible for the direct estrogen actions on the monkey hippocampus, regulating glial response after ischemia.     

Development of somatostatin-like immunoreactivity in the hippocampal formation of normal and reeler mice

Using antisera directed against somatostatin-28 or somatostatin-28(1-12), the development of somatostatin-like immunoreactivity (SS-LI) was examined in the hippocampal formation of normal and reeler mice. As early as postnatal day 5, SS-labeled neurons exhibit the adult pattern of distribution in the normal hippocampal formation, these neurons being situated predominantly in the stratum oriens of the hippocampus and the hilus of the dentate gyrus. In contrast, SS-labeled neurons in the reeler hippocampal formation are dispersed throughout the various layers, reflecting the disrupted laminar organization of the hippocampus in this mutant. In both the normal and reeler hippocampal formation, SS-labeled fibers are most abundant in the stratum lacunosum moleculare. However, in the reeler, there appears to be increase in the density of SS-LI fibers, not only in the stratum lacunosum moleculare but also in the stratum oriens, stratum radiatum and pyramidal cell layer of the hippocampus.     

Short-term changes of parvalbumin and calbindin immunoreactivity in the rat hippocampus following cerebral ischemia

The calcium-binding proteins, parvalbumin (PV) and calbindin (CaBP), were used as immunocytochemical markers for two different interneuron populations in the rat hippocampus shortly after transient cerebral ischemia. Besides in interneurons, CaBP immunoreactivity (-i) is located in hippocampal CA1 pyramidal cells and dentate granule cells. Shortly after ischemia, the PV-i and CaBP-i were unchanged but, around the 4th postischemic day, PV-i disappeared from somata and fibers located in CA1, CA3c, and the dentate hilus. Terminal PV-i was unchanged. Within days, the PV-i gradually reappeared, first in somata and then in fibers. The transient loss of PV-i was, on a time scale, closely accompanied by a permanent loss of CaBP-i in CA1 pyramidal cells. CaBP-i in interneurons was unchanged. In order to examine the effect of an increased intracellular calcium concentration on the PV-i and CaBP-i, the calcium ionophore A23187 was stereotaxically injected into CA1. In rats killed 30 min later and processed for PV-i and CaBP-i, both PV-i and CaBP-i had disappeared around the A23187 injection sites. Based on this observation and the changes observed after ischemia, it is suggested that the hippocampal PV-i interneurons suffer from a delayed and reversible calcium accumulation in the days after ischemia. Concomitantly, there could be a decreased synthesis or increased destruction of PV after ischemia.     

Long-term study of dendritic spines from hippocampal CA1 pyramidal cells, after neuroprotective melatonin treatment following global cerebral ischemia in rats

Melatonin reduces pyramidal neuronal death in the hippocampus and prevents the impairment of place learning and memory in the Morris water maze, otherwise occurring following global cerebral ischemia. The cytoarchitectonic characteristics of the hippocampal CA1 remaining pyramidal neurons in brains of rats submitted 120 days earlier to acute global cerebral ischemia (15-min four vessel occlusion, and melatonin 10mg/(kg h 6h), i.v. or vehicle administration) were compared to those of intact control rats in order to gain information concerning the neural substrate underlying preservation of hippocampal functioning. Hippocampi were processed according to a modification of the Golgi method. Dendritic bifurcations from pyramidal neurons in both the oriens-alveus and the striatum radiatum; as well as spine density and proportions of thin, stubby, mushroom-shaped, wide, ramified, and double spines in a 50 microm length segment of an oblique dendrite branching from the apical dendrite of the hippocampal CA1 remaining pyramidal neurons were evaluated. No impregnated CA1 pyramidal neurons were found in the ischemic-vehicle-treated rats. CA1 pyramidal neurons from ischemic-melatonin-treated rats showed stick-like and less ramified dendrites than those seen in intact control neurons. In addition, lesser density of spines, lower proportional density of thin spines, and higher proportional density of mushroom spines were counted in ischemic-melatonin-treated animals than those in the sinuously branched dendrites of the intact control group. These cytoarchitectural arrangements seem to be compatible with place learning and memory functions long after ischemia and melatonin neuroprotection.     

Post-ischemic synaptic plasticity in the rat hippocampus after long-term survival: histochemical and autoradiographic study.

The hippocampus provides a suitable area in the brain for the analysis of neuronal plasticity after application of a selective lesioning technique. Using histochemistry and autoradiography, we studied synaptic reorganization in the rat hippocampus with selective CA1 pyramidal cell lesioning caused by transient forebrain ischemia after long-term survival. An autoradiographic study was performed on second messenger systems ([3H]inositol 1,4,5-trisphosphate, [3H]forskolin and [3H]phorbol 12,13-dibutyrate binding). One-hundred days after ischemia, depletion of CA1 pyramidal cells and marked shrinkage of the CA1 subfield was noted in spite of unaltered thickness of the CA3 band and of the dentate molecular layers. Although neuronal density in the CA3 region of animals killed seven days after ischemia was not different from the normal group, 78% of animals showed neuronal loss of 30-50% in the stratum pyramidale of the CA3b 100 days after recirculation. Sixty-seven per cent of animals exhibited supragranular mossy fiber sprouting in the dentate gyrus. However, CA3 neuronal loss did not correlate with mossy fiber sprouting. Succinic dehydrogenase was depleted in the CA1 100 days after ischemia, and animals with CA3 damage showed a reduction of succinic dehydrogenase activity in the CA3. In contrast to the unaltered acetylcholinesterase in the animals killed seven days after ischemia, high density bands of acetylcholinesterase activity in the stratum pyramidale of the CA1 were found to be broadened 100 days after ischemia. In the CA1 subfield, subnormal activity of [3H]phorbol 12,13-dibutyrate and [3H]forskolin binding were observed in spite of the depleted [3H]inositol 1,4,5-triphosphate binding. [3H]Forskolin binding in the hilus had increased by 62% 100 days after ischemia, although binding in the stratum lucidum of the CA3 and in the stratum moleculare of the dentate gyrus was unaltered. However, no visible supragranular increase in [3H]forskolin binding was observed. These results indicate that long-term survival after CA1 pyramidal cell depletion caused by transient forebrain ischemia induced the modulation of neuronal activity and synaptic rearrangements in the whole hippocampal formation.     

Ischemia changes the coexpression of somatostatin and neuropeptide Y in hippocampal interneurons

Transient cerebral ischemia causes extensive cell death in hippocampal CA1 pyramidal cells and selective loss of interneurons in the dentate hilus. Many hippocampal interneurons can be classified by their contents of somatostatin (SS) and/or neuropeptide Y (NPY). Following ischemia in the rat, most of the NPY immunoreactivity is permanently lost in hippocampus. Furthermore, SS interneurons in the dentate hilus die, whereas CA1 interneurons survive and their expression of SS mRNA and peptide returns to preischemic levels within 16 days after ischemia. We have addressed the following questions: (1) Does the loss of NPY involve a specific downregulation in surviving CA1 interneurons that preischemically expressed both SS and NPY? (2) Can the subpopulation of dying interneurons in hilus be identified from their preischemic coexpression of SS and NPY? We investigated the coexpression of SS mRNA and NPY peptide using combined in situ hybridization and immunocytochemistry. Cells containing one or both markers were counted in control sections and sections taken 2–16 days after ischemia from the hippocampal formation. In CA1, a decrease in the number of neurons containing NPY alone as well as a decrease in the number of neurons coexpressing NPY and SS was observed, whereas the number of neurons containing SS alone increased 16 days after ischemia. We conclude that neurons coexpressing SS and NPY before ischemia added to the number of neurons containing SS alone after ischemia, because NPY expression was selectively downregulated in the coexpressing population. In hilus, we demonstrated both survival and ischemic cell death of neurons expressing either SS, NPY or both, indicating that hilar interneurons dying from ischemia cannot unequivocally be identified from their preischemic colocalization of SS and NPY.     

Impairment of protein ubiquitination may cause delayed neuronal death

The hippocampus is a brain structure specifically vulnerable to short periods of transient cerebral ischemia, and which displays delayed neuronal necrosis. Protein ubiquitination is a posttranslational modification of proteins and an important factor in heat shock response and a regulator of ATP-dependent protein degradation. Using affinity purified antibodies against ubiquitin and ubiquitin-protein conjugates we have found that the ubiquitin immunoreactivity (UIR), normally present in all neurons of the hippocampus, disappears in the early recirculation period following cerebral ischemia from all hippocampal cells except the interneurons. Later UIR reappears in the different hippocampal regions over a 72 h period in the following order: granule cells-CA3 pyramidal cells-CA2 pyramidal cells. This is the inverse order of sensitivity of these cells to ischemia. The UIR never recovers in the CA1 pyramidal neurons where a 95% neuronal necrosis is seen following three days of recovery. We propose that the loss of UIR in the pyramidal neurons in the CA1 region signifies a persistent impairment of protein ubiquitination, and thus a change in the turnover of structural and regulatory proteins, which could be an essential part of the mechanism of slow neuronal death following cerebral ischemia.     

Changes in excitatory and inhibitory circuits of the rat hippocampus 12-14 months after complete forebrain ischemia.

Changes in interneuron distribution and excitatory connectivity have been investigated in animals which had survived 12-14 months after complete forebrain ischemia, induced by four-vessel occlusion. Anterograde tracing with Phaseolus vulgaris leucoagglutinin revealed massive Schaffer collateral input even to those regions of the CA1 subfield where hardly any surviving pyramidal cells were found. Boutons of these Schaffer collaterals formed conventional synaptic contacts on dendritic spines and shafts, many of which likely belong to interneurons. Mossy fibres survived the ischemic challenge, however, large mossy terminals showed altered morphology, namely, the number of filopodiae on these terminals decreased significantly. The entorhinal input to the hippocampus did not show any morphological alterations. The distribution of interneurons was investigated by neurochemical markers known to label functionally distinct GABAergic cell populations. In the hilus, spiny interneurons showed a profound decrease in number. This phenomenon was not as obvious in CA3, but the spiny metabotropic glutamate receptor 1alpha-positive non-pyramidal cells, some of which contain calretinin or substance P receptor, disappeared from stratum lucidum of this area. In the CA1 region, somatostatin immunoreactivity disappeared from stratum oriens/lacunosum-moleculare-associated cells, while in metabotropic glutamate receptor 1alpha-stained sections these cells seemed unaffected in number. Other interneurons did not show an obvious decrease in number. In stratum radiatum of the CA1 subfield, some interneuron types had altered morphology: the substance P receptor-positive dendrites lost their characteristic radial orientation, and the metabotropic glutamate receptor 1alpha-expressing cells became extremely spiny. The loss of inhibitory interneurons at the first two stages of the trisynaptic loop coupled with a well-preserved excitatory connectivity among the subfields suggests that hyperexcitability in the surviving dentate gyrus and CA3 may persist even a year after the ischemic impact. The dorsal CA1 region is lost; nevertheless hyperactivity, if it occurs, may have a route to leave the hippocampus via the longitudinally extensive axon collaterals of CA3 pyramidal cells, which may activate the subiculum and entorhinal cortex with a relay in the surviving ventral hippocampal CA1 region.