Microglial and Astrocytic Cell Responses in the Rat Hippocampus after an Intracerebroventricular Kainic Acid Injection

After central nervous system injury activated microglial cells and reactive astrocytes secrete neurotrophic factors which may provide an environment conducive to axonal sprouting. The present study has used a unilateral intracerebroventricular (ICV) injection of kainic acid (KA) to produce a lesion of the CA3 pyramidal neurons in the rat hippocampus. The time course of the microglial and astrocytic response was studied throughout a 3-month period using an antibody to proliferating cell nuclear antigen to identify proliferating cells as well as OX-42 and GFAP antibodies to identify the activated microglia and reactive astrocytes, respectively. There was no proliferation of reactive astrocytes whereas activated microglial cells continued to proliferate throughout the duration of the study. During the first month there were some activated microglial cells in the CA1 field and in the fascia dentata but this was short-lived in comparison to the persistance of activated microglia and reactive astrocytes in the CA3 field which were still present 3 months after the initial injection. The discussion attempts to correlate this ipsilateral microglial and astrocytic response with the bilateral mossy fiber axonal sprouting, which occurs in the dentate gyrus after a unilateral ICV injection of KA. The discussion concludes that the two events, the microglial and astrocytic response and the mossy fiber sprouting, are not directly related since the contralateral sprouting occurs in the absence of any astrocytic or microglial response.     

Microglial MHC antigen expression after ischemic and kainic acid lesions of the adult rat hippocampus

By taking advantage of the specific neuronal and connective organization of the hippocampus and the different susceptibility of hippocampal neurons to transient cerebral ischemia or intraventricular injections of kainic acid (KA), we examined the microglial reactions to different types of neuronal injury. In all areas with neuronal or axonal degeneration, the microglial cells reacted by specific degeneration-related morphological transformations and expression of class I major histocompatibility complex (MHC) antigen. Subpopulations of microglial cells also expressed class II MHC antigen and leukocyte common antigen (LCA) in relation to (1) degenerating nerve cell bodies in the dentate hilus and the CA1 and CA3 pyramidal cell layers, (2) postischemic degeneration of dendrites in the stratum radiatum of CA1, and (3) combined dendritic and axonal degeneration in the stratum radiatum of the KA-lesioned CA3. MHC II and LCA expression was not observed in relation to degeneration of the CA3-derived Schaffer collaterals in CA1 after KA-induced CA3 lesions. In the case of ischemia the degeneration-related reactions were preceded by an early, generalized microglial reaction, which also included areas without subsequent signs of neural degeneration. This reaction, which was transient and characterized by subtle morphological changes and induction of class I MHC antigen only, was presumably triggered by a general postischemic pertubation of the cerebral microenvironment, and not by actual neural degeneration. In conclusion, we found that microglial expression of class I MHC antigen was a sensitive marker of both the general pertubation after ischemia and axonal degeneration distant from the areas of actual nerve cell death. Induction of microglial LCA and class II MHC antigen expression, together with protracted expression of class I, was, characteristic of a protracted reaction, only found in areas with degeneration of nerve cell bodies and dendrites.     

Microglial and astrocytic reactions prior to onset of thalamic cell death after traumatic lesion of the rat sensorimotor cortex

The temporospatial relationship between microglial and astrocytic reactions and delayed thalamic cell death was examined 1–7 days following a traumatic cold lesion of the rat sensorimotor cortex using immunocytochemistry in combination with terminal deoxynucleotidyltransferase-mediated biotinylated dUTP nick end labeling (TUNEL) of nuclear DNA fragmentation. No or only occasional TUNEL-positive cells were found in the thalamic relay nuclei up to 3 days after trauma. After 7 days, on the other hand, a considerable number of TUNEL-positive cells were seen in the ventrobasal, the ventrolateral and posterior thalamic nuclei. Already 3 days after trauma, i.e., before cell injury was detectable, many protoplasmic astrocytes, which were reactive for glial fibrillary acidic protein, and ramified microglia, which were positive for complement receptor type 3b (CR3b) but negative for major histocompatibility complex (MHC) class II antigen, were noticed in the thalamus. The number of labeled astro- and microglia further increased after 7 days, when DNA fragmentation became evident. At this time, the morphology of microglia shifted towards bushy and rod-like cells, and microglia became also reactive for MHC class II antigen. Clusters of CR3b- and MHC class II-positive microglia were found in the ventrobasal thalamus. The present findings demonstrate that trauma-induced microglial and astrocytic reactions appear in the thalamus prior the onset of cell damage. Received: 25 February 1999 / Accepted: 22 June 1999     

Kainic acid activates transient expression of tenascin-C in the adult rat hippocampus

Kainic acid-induced limbic seizures enhance expression of tenascin-C (TN) in the hippocampus of adult rats. TN mRNA was detectable by in situ hybridization in many granule cells in the dentate gyrus 4.5 hr after kainic acid injection but not in saline-injected animals (controls) or in animals killed 2 or 24 hr after injection. Thirty days after kainic acid injection, TN mRNA was detectable only in pyramidal cells of CA3 and CA1. At the protein level, TN was detectable by immunocytochemistry in control animals in the strata oriens and lacunosum moleculare of CA1, in the molecular layer, and within a narrow area at the inner surface of the granule cell layer in the dentate gyrus. Twenty-four hours after kainic acid injection, TN immunoreactivity was enhanced in these areas and throughout the granule cell layer. Thirty days after kainic acid injection, TN immunoreactivity was downregulated in these areas, while it was prominent in the stratum oriens and in clusters of immunoreactivity in the stratum lucidum of CA3. Western blot analysis of the hippocampus showed a peak of TN expression 24 hr after kainic acid injection. These observations show that TN expression is upregulated in predominantly neuronal cells already by 4.5 hr after kainic acid injection, coincident with activation of granule cells and sprouting of axon terminals, whereas the remaining TN expression 30 days after injection relates to pyramidal cells in CA1 and CA3, coincident with an astroglial response, as marked by a strong expression of glial fibrillary acidic protein. © 1996 Wiley-Liss, Inc.     

Differential cellular response after glutamate analog hippocampal damage

The tissue response after brain damage implicates the cellular “activation” of astrocytes and microglia. This glial response is referred as reactive gliosis. Using immunohistochemical markers, we have analyzed the neuronal and glial response to some neurotoxic-induced lesions. We have compared the effects of two glutamate analogs, AMPA and kainic acid, with those of traumatic injury. Our data showed that the time-course of appearance, the relative contribution of and the behavior of reactive astrocytes and microglial cells were clearly different after AMPA or kainic acid administration. The immunoreactivity associated with microglia response, with respect to the immunoreactivity associated with reactive astrocytes, was higher after AMPA damage than after kainic acid treatment. In both cases, however, glial cells were more abundant than after traumatic lesions. Interestingly, the CA1 pyramidal neurons affected by AMPA and some cortical neurons affected by traumatic injury responded with an overexpression of amyloid precursor protein, whereas no neuronal response was detected after the kainic acid treatment. Our data suggest that the gliotic response is highly specific to the type of insult and heterogeneous depending on the brain area affected. © 1996 Wiley-Liss, Inc.     

Characterization of microglial reaction after middle cerebral artery occlusion in rat brain

We have studied the microglial reaction that accompanies cortical infarction induced by middle cerebral artery occlusion (MCAO). Lectin histochemistry with the B₄-isolectin from Griffonia simplicifoliaas well as immunocytochemistry with a panel of monoclonal antibodies directed against major histocompatibility complex (MHC) and lymphocytic antigens were performed. Principal attention was focused on neocortical and thalamic regions, representative of primary and secondary ischemic damage, respectively. With the lectin procedure, activated microglial cells were abundant in the neocortex 24 hours after MCAO. In contrast, microglial activation in the thalamus was not apparent until day 2 after MCAO. On day 5, MHC class II antigen was expressed by reactive microglia in fiber tracts traversing the striatum, but was absent from activated microglia in the primary cortical infarction area. MHC class I and lymphocytic antigens were expressed differentially on microglia with class I antigens appearing early and lymphocytic antigens appearing late in the time course after focal ischemia. The findings are compatible with previous studies during global ischemia and confirm the early activation and the progressive nature of immunomolecule expression on activated microglia after an ischemic insult. In addition to neocortical and thalamic sites, our results showed an early microglial activation to be present also in forebrain regions outside of the middle cerebral artery (MCA) territory, such as the contralateral cortex and hippocampus. A unilateral microglial reaction was also detectable after long-term survival (≥4 weeks) in the pyramidal tracts, as well as in the corticospinal tracts at cervical but not lumbar spinal cord levels. Ischemia-induced neuronal damage, as evaluated by Nissl staining, was found only in cortical and thalamic regions. We conclude that the demonstration of reactive microglia indicates not only imminent ischemic neuronal damage within MCA territory but can also delineate extra-focal disturbances, possibly reflecting subtle and transitory changes in neuronal activity. © 1993 Wiley-Liss, Inc.     

Expression of Heat Shock Protein-70 and Limbic Seizure-induced Neuronal Death in the Rat Brain

The effect of MK-801, a non-competitive N-methyl-D-aspartate (NMDA) antagonist, on the kainic acid-induced expression of the inducible heat shock protein 70 kDa (HSP70) and on neuronal death in the rat hippocampus was investigated. HSP70 is expressed in ?80% of the pyramidal neurons in the CA1 field 1 day after kainic acid injection. The majority of these HSP70-immunopositive neurons exhibited swelling and a hollow appearance in the perikaryon, indicating that they had been injured following kainic acid-elicited limbic seizures. Four days after administration of kainic acid, 87% of the pyramidal neurons in the CA1 field were dead. When a single dose of MK-801 was administered 1 h before kainic acid injection, the number of rats suffering with seizures was reduced, the severity of limbic seizures was attenuated and seizure onset was delayed. Neither HSP70 expression on day 1 nor neuronal loss on day 4 in the CA1 pyramidal cell layer was observed in these animals. A considerable number of HSP70-immunopositive neurons was detected in the dentate hilus, however, and somewhat fewer in the CA3a and CA3c subfields on day 1. Severe neuronal damage in these regions followed on day 4. Interestingly, little HSP70 expression or neuronal loss was observed in the CA3b subfield in these same animals. When a single dose of MK-801 was given 4 h after kainic acid treatment, HSP70 expression was partially blocked; 18% of neurons expressed HSP70 on day 1 and 37% on day 4 in CA1 pyramidal neurons in comparison to the kainic acid controls. About 50% neuronal death was detected in the CA1 pyramidal cell layer 4 days after kainic acid treatment followed by MK-801. When the animals were treated with MK-801 4 h after kainic acid treatment followed by additional daily administration for 3 days, a negligible number of pyramidal neurons expressed HSP70, and the survival of pyramidal cells was significantly increased in the CA1 field. Limbic seizure-induced HSP70 expression not only indicates neuronal injury in the pyramidal cell layer of the hippocampus but also predicts delayed neuronal death, at least in the case of the CA1 field of animals that suffered stage IV—V seizures.     

The selective cyclooxygenase-2 inhibitor rofecoxib reduces kainate-induced cell death in the rat hippocampus

Treatment of male Sprague-Dawley rats with kainic acid (10 mg/kg, i.p.) triggered limbic seizures in 60% of the animals starting within 30 min and lasting for about 6 h. Cyclooxygenase-2 (COX-2) mRNA was strongly induced in the pyramidal cells of the hippocampus, in the amygdala and the piriform cortex after 8 h, as shown by in situ hybridization, and returned to control levels after 72 h. At this time marked cell loss occurred in the CA1-CA3 areas of the hippocampus. We hypothesize that rofecoxib, a selective COX-2 inhibitor, might abbreviate the late neurotoxicity, possibly associated with COX-2 induction. Animals which developed seizures were treated for 3 days with rofecoxib (10 mg/kg, i.p., n = 12) starting 6 or 8 h after kainic acid injection. Histological staining of viable cells confirmed that rofecoxib treatment selectively diminished cell loss in the hippocampus. The TdT-mediated dUTP nick end labelling (TUNEL) technique was used to estimate delayed cell death. Abundant TUNEL-positive cells were detected in seizure rats 72 h after kainic acid injection in pyramidal cells of the hippocampus (CA1-CA3), in cells of the thalamus, the amygdala and the piriform cortex. Treatment with rofecoxib selectively and significantly (P < 0.05) attenuated the number of TUNEL-positive cells in the hippocampus, whereas the cells of the thalamus, amygdala and piriform cortex were not protected. Therefore we conclude that COX-2 might contribute to cell death of pyramidal cells of the hippocampus as a consequence of limbic seizures.     

Post- and presynaptic lesions in the CA1 region of hippocampus: Effect on [3H]forskolin and [3H]phorboldibutyrate ester binding

Summary The effect of transient cerebral ischemia and intraventricular injection of kainic acid on adenylate cyclase and protein kinase C as labeled by [³H]forskolin ([³H]FOR) and [³H]phorboldibutyrate ester ([³H]PDBU) in several rat brain microregions was investigated in a quantitative autoradiographic study. Four days after transient four vessel occlusion a 80% loss of [³H]FOR and a 35% loss of [³H]PDBU binding could be measured in the CA1 stratum radiatum of operated Wistar rats as compared to control rats. Four days after intraventricular injection of kainic acid only a marginal loss of [³H]FOR and a 30% increase of [³H]PDBU binding was seen in the CA1 stratum radiatum while in the CA3 stratum lucidum and radiatum respectively a 30% loss of [³H]FOR and no significant change in [³H]PDBU binding was observed. As transient cerebral ischemia and intraventricular kainic acid injection are depleting the hippocampal CA1 region of CA1 pyramidal cells and axons of CA3 pyramidal cells respectively in rat brain, these findings strongly suggest that both adenylate cyclase and protein kinase C are localized in CA1 pyramidal cells of rat hippocampus.Part of this study has been presented at the 16th C.I.N.P. Congress, Munich, August 15–19, 1988.     

MHC Class II expression by microglia in tetanus toxin–induced experimental epilepsy in the rat

Minute amounts of tetanus toxin injected into the hippocampus of rats results in an epileptiform syndrome. When the toxin injection is made unilaterally or bilaterally into the ventral hippocampus, about one–third of animals with seizures show bilateral neuronal loss in dorsal CA1 of the hippocampus after 1 week. In animals with seizures, microglia in hippocampus are found to be activated. The present work shows that during the acute phase, microglia in the substantia nigra become activated and express MHC class II antigens in the majority of animals with seizures. After the animals have recovered from the acute phase at 8 weeks, the MHC class II expression has largely disappeared from the substantia nigra but MHC class II–expressing microglia are found in the dorsal hippocampus of those rats with loss of cells from CA1. These results show that microglia are responsive to abnormal electrical activity in the central nervous system in the absence of degenerative changes. Further studies are required to determine how microglia may contribute to the neuropathology of epilepsy.     

Dentate granule neuron apoptosis and glia activation in murine hippocampus induced by trimethyltin exposure

We investigated the effect of trimethyltin (TMT), a well-known neurotoxicant, on murine hippocampal neurons and glial cells. Three days following intraperitoneal (i.p.) injection of TMT into 1-month-old Balb/c mice at a dose of 2.5 mg/kg body weight we detected damage of the dentate gyrus granular neurons. The dying cells displayed chromatin condensation and internucleosomal DNA fragmentation, which are the most characteristic features of apoptosis. To study, if prolyl oligopeptidase is engaged in neuronal apoptosis following TMT administration, we pretreated mice with the specific inhibitor--Fmoc-Pro-ProCN in doses of 5 and 10 mg/kg body weight (i.p. injection). Three days following injection we did not observe any attenuation of neurotoxic damage, regardless of inhibitor dose, indicating the lack of prolyl oligopeptidase contribution to neuronal injury caused by TMT. The neurodegeneration was associated with reactive astrogliosis in whole hippocampus, but particularly in injured dentate gyrus. The reactive astrocytes showed an increased nerve growth factor (NGF) expression in ventral as well as dorsal hippocampal parts. NGF immunoreactivity was also augmented in neurons of CA3/CA4 areas, which were almost totally spared after TMT intoxication. It suggested a role for this neurotrophin in protection of pyramidal cells from loss of connection between CA3/CA4 and dentate gyrus fields. The granule neurons' death was accompanied by increased histochemical staining with isolectin B4, a marker of microglia, in the region of neurodegeneration. The microglial cells displayed ramified and ameboid morphology, characteristic of their reactive forms. Activated microglia were the main source of interleukin 1beta (IL-1beta). It is possible that this cytokine may participate in neurodegeneration of granule cells. Alternatively, IL-1beta elaborated by microglia could play a role in increasing NGF expression, both in astroglia and in CA3/CA4 neurons.     

Time-dependent changes in distribution of basic fibroblast growth factor immunoreactive cells in hippocampus after kainic acid injection in rat pups

Five-day-old Wistar albino rats were injected with kainic acid (KA) or saline i.p. to investigate time-dependent alterations in morphology and number of basic fibroblast growth factor (bFGF) immunoreactive (-ir) astrocytes and neurons in hippocampus at 15, 30, and 90 days after the injections. Sections were stained with cresyl violet for morphological evaluation and bFGF immunohistochemistry was used for quantitative evaluation of bFGF-ir cell density. Fifteen days after KA injection, there was gliosis but no neuronal loss although disorganization in CA1, CA3, CA4 pyramidal layers and neuronal loss were evident 30 and 90 days after the injection. KA injected rats demonstrated significantly increased number of bFGF-ir astrocytes throughout the hippocampus and pyramidal neurons in CA2 after 15 days and decreased number of bFGF-ir cells after 30 and 90 days. The decrease in the number of bFGF-ir astroglia and neurons in long term after KA injection may indicate a decrease in the production of bFGF and/or number of bFGF-ir cells suggesting that protective effects of bFGF may be altered during epileptogenesis in hippocampus.     

Time-dependent changes in distribution of basic fibroblast growth factor immunoreactive cells in rat hippocampus after status epilepticus

OBJECTIVE: In this study, we aimed to examine time-dependent morphologic changes and quantitative alterations in the density of basic fibroblast growth factor (bFGF)-immunoreactive (ir) astrocytes and CA2 pyramidal neurons in dorsal hippocampus of rats after status epilepticus (SE) induced by kainic acid (KA) injection. METHODS: Wistar albino rats were injected with saline or KA i.p. to investigate time-dependent alterations in morphology and the number of bFGF-ir astrocytes and neurons in the dorsal hippocampus 15, 30 and 90 days after KA injection. RESULTS: Fifteen days after KA injection, gliosis was present throughout the hippocampus and neuronal loss was evident in CA1 and CA3 regions, which was more severe after 30 and 90 days. KA-injected rats demonstrated significantly increased number of both bFGF-ir astrocytes throughout the hippocampus and pyramidal neurons in CA2 after 15 days and decreased number after 30 and 90 days. CONCLUSION: The decrease in the number of bFGF-ir astroglia and neurons in long term after KA injection may indicate a decrease in the production of bFGF and/or number of bFGF-ir cells, suggesting that protective effects of bFGF might be altered during epileptogenesis in the hippocampus.     

Expression of non-angiotensin II [I]CGP 42112 binding sites on activated microglia after kainic acid, induced neurodegeneration

[¹²⁵I]CGP 42112, first developed to identify angiotensin II receptor subtype 2 (AT₂), was recently shown to bind to a novel non-angiotensin binding site in injured rat brain tissue. We addressed the question whether non-angiotensin [¹²⁵I]CGP 42112 binding appears after kainic acid induced hippocampal neurodegeneration, a process of neuronal cell death at a distance from the toxin injection site. After intraventricular kainic acid injection, we found non-angiotensin [¹²⁵I]CGP 42112 binding in the hippocampal areas CA3 (4 and 14 days after injection), CA1 and CA4 and the subiculum (14 days after injection). In addition, 14 days after kainic acid injection, [¹²⁵I]CGP 42112 binding was found in 50% of the animals, in the thalamus, amygdala and piriform cortex, areas receiving projections from the hippocampus and suffering kainic acid induced delayed neurodegenaration. The loss of neurons in these regions was accompanied by an accumulation of activated microglia as demonstrated by immunostaining with the specific antibodies OX-42 and ED1. The time course and regional pattern of OX-42/ED1 positive immunostaining was identical with the appearance and distribution of the non-angiotensin [¹²⁵I]CGP 42112 binding site. The non-angiotensin [¹²⁵I]CGP 42112 binding was not detected in brain regions unaffected by kainic acid injection. Our findings indicate the expression of a novel [¹²⁵I]CGP 42112 binding site on activated microglia. This site appears at a distance from the lesion and may be of importance in the process of neuronal death and brain tissue repair.     

Endogenous BDNF Protein Is Increased in Adult Rat Hippocampus after a Kainic Acid Induced Excitotoxic Insult but Exogenous BDNF Is Not Neuroprotective

Systemic administration of the excitotoxin kainic acid to adult rats results in a well defined pattern of loss of the CA1 and CA3 pyramidal neurons of the hippocampus. Prior to this neuronal loss, brain-derived neurotrophic factor (BDNF) mRNA is substantially increased. We show here that BDNF protein is increased after excitotoxic insult in specific areas of the hippocampus, reaching maximal levels 24 h after the insult. BDNF protein levels in the hippocampus increase in direct relation to the severity of seizure. Up to 7 days after injection of kainic acid, levels of full-length Trk B protein were unchanged, whereas levels of truncated TrkB protein were significantly increased by 12 h. To determine whether elevations in BDNF protein levels are potentially beneficial to hippocampal neurons exposed to an excitotoxic stress, we infused exogenous BDNF prior to and during the period of neuronal death caused by kainic acid. We find that administration of high levels of exogenous BDNF does not affect severity of seizure, but does in fact, exacerbate the injury caused by kainic acid, specifically to CA3 pyramidal neurons. Although there was a trend toward sparing of CA1 pyramidal neurons on the side infused with BDNF, this was not significant. In the same paradigm, infusion of exogenous NT-3 had no effect.     

Changes in the expression of astroglial and microglial markers in the hippocampus of rats adapted to chronic stress and the effects of panthenol

We studied the morphological changes and expression of the astroglial and microglial markers in the sensorimotor cortex and hippocampus of rats that were subjected to chronic stress. Chronic neurotization was associated with the appearance of a large number of damaged neurons in layer V of the sensorimotor cortex and the pyramidal layer of the hippocampus. These changes were considerably expressed in the CA3 field. A neurosis-like state in rats was accompanied by an increased expression of microglial markers in the hippocampus and this effect was evident even 1 month after the end of stress. The expression of the astroglial marker GFAP decreased in the CA3 hippocampal field. Treatment of animals with the pantothenic acid derivative panthenol stabilized the glial response to chronic stress; however, its effects were not long-lasting and did not prevent activation of microglia during the period after stress.     

Temporal lobe epilepsy of the rat: differential expression of mRNAs of chromogranin B, secretogranin II, synaptin/synaptophysin and p65 in subfield of the hippocampus.

We have investigated by in situ hybridization changes in the content of mRNAs encoding for chromogranin B, secretogranin II, synaptin/synaptophysin and p65 after kainic acid-induced seizures and pentylenetetrazol kindling. Kainic acid seizures resulted in marked but transient increases in secretogranin II mRNA concentrations in the granule cell layer and throughout the pyramidal cell layers of the hippocampus (by 100-500%) as well as in various areas of the cerebral cortex (by up to 900%) and the thalamus (up to 300%) 12 h after injection of the toxin. Chromogranin B mRNA concentrations were persistently increased in granule cells (but not in pyramidal cells) of the hippocampus (suprapyramidal blade, 450%) and in cortical areas (250%) at all time intervals after kainic acid injection (12 h to 60 days). Accordingly chromogranin B immunoreactivity was enhanced in the terminal field of mossy fibers and in the inner part of the molecular layer 30 days after kainic acid. Secretogranin II immunoreactivity was also markedly increased in CA1, the paraventricular thalamic nucleus and in the central amygdala. In rats kindled with pentylenetetrazol only chromogranin B (by 200%) but not secretogranin II mRNA was increased in dentate granule cells. In contrast to the mRNAs of these secretory proteins concentrations of mRNAs encoding synaptin/synaptophysin and p65, two membrane proteins of synaptic vesicles, were not altered in any of these brain structures. These data demonstrate that in brain the biosynthesis of chromogranin B and secretogranin II is regulated like that of neuropeptides which is consistent with a role of these secretory polypeptides as precursors of functional peptides. Activation of neurons induces an increased synthesis of neuropeptides but not a concomitant synthesis of membrane proteins of synaptic vesicle. This might lead to an increased quantal content available for transmission.     

Long-term potentiation in Schaffer collateral and commissural systems of the hippocampus: In vitro study in rats pretreated with kainic acid

Experiments were carried out on hippocampal slices from rats pretreated with a unilateral intraventricular injection of kainic acid. The kainic acid produced a lesion of the CA3 area in the hippocampus ipsilateral to the site of injection. The resulting neuroanatomically modified hippocampal slices allowed the independent study of the Schaffer collateral and commissural afferents that synapse onto CA1 pyramidal cells. The Schaffer collateral and commissural afferents were both found to independently support long-term potentiation (LTP), and the properties of the LTP were similar to those observed in slices cut from unlesioned hippocampi.     

Temporal profiles of the in vitro phosphorylation rate and immunocontent of glial fibrillary acidic protein (GFAP) after kainic acid-induced lesions in area CA1 of the rat hippocampus: demonstration of a novel phosphoprotein associated with gliosis

The in vitro phosphorylation rate and immunocontent of glial fibrillary acidic protein was studied in slices of area CA1 of the rat hippocampus after stereotaxic injection of 1 nmol of kainic acid. For controls the contralateral hippocampus was injected with saline. Hippocampal tissue was incubated with [³²P]phosphate and analysed by two-dimensional electrophoresis for phosphorylation rate and by immunoblotting for immunocontent. Both these parameters decreased during the first 4 days after injection and then started to increase at 10 days and continued to increase until at least 84 days. Except for a small excess of phosphorylation rate at 28 days, the relationship between immunocontent and in vitro phosphorylation rate of glial fibrillary acidic protein remained constant, indicating that the reactive gliosis was not associated with hypo- or a major hyperphosphorylation of this protein. Histology showed a pronounced loss of CA1 pyramidal cells 1 day after injection. At 28 days after injection the pyramidal cells had disappeared and only a few abnormal neurones were present. In contrast, immunocytochemistry after 28 days showed a marked increase in astrocytes reacting positive to the antibody in the strata radiatum and lacunosum moleculare. Besides glial fibrillary acidic protein the expression of several other proteins was upregulated as a result of the injection of kainic acid. These included phosphovimentin and an unknown phosphoprotein designated pp25 which co-migrated on 2-D gels with a prominent phosphoprotein expressed in primary cultures of astrocytes. Pp25 was expressed in lesioned tissue more frequently than phosphovimentin and with a time course that started earlier. Of particular interest was the expression of pp25 in the contralateral saline-injected hippocampus 1 day after injection of kainic acid. It is possible that pp25 will prove to be a sensitive marker of gliosis.