The Distribution of β-Amyloid Precursor Protein in Rat Cortex after Systemic Kainate-Induced Seizures

EXPERIMENTAL NEUROLOGY ARTICLE NO.

147, 361–376 (1997)

EN976622

The Distribution of b-Amyloid Precursor Protein in Rat Cortex after Systemic Kainate-Induced Seizures Shai Shoham1 and Richard P. Ebstein Shapiro Molecular Neurobiology Laboratory, S. Herzog Memorial Hospital, P.O. Box 35300, Jerusalem 91351, Israel

INTRODUCTION In the current study we employed immunohistochemical techniques to identify neuronal and glial cells in specific brain areas that modulate b-amyloid precursor protein (bAPP) synthesis following kainateinduced seizures. In addition, antibodies directed against the FOS protein, which is generated by activation of the immediate early gene c-fos and is temporally associated with ongoing seizure activity, were used to identify transneuronal pathways activated after kainate-induced seizures (KIS). It was therefore possible to correlate the appearance of activated neuronal pathways identified by FOS-like immunoreactivity (LI) and bAPP-LI in alternate sections. In addition, we employed immunohistochemical procedures to characterize morphological changes in neuronal and glial cells following kainate-induced seizures in both young and adult rats. Our results demonstrate a specific pattern of FOS-LI induced by kainate injection. In older animals FOS-LI spreads out from limbic cortical regions, including the piriform and entorhinal cortex, to other cortical regions, including the parietal and somatosensory cortices. Seizures were associated with decrease in neuronal bAPP-LI in both young and adult rats, whereas glial bAPP-LI markedly increased. The increase in bAPP-LI glia was far more extensive in adult than in young rats and the anatomical distribution of bAPP-LI glia was grossly correlated with FOSLI. The spread of bAPP-LI follows seizure-activated transsynaptic pathways. It is likely that the sequence of events following kainate injection is initially triggered by c-fos gene expression, which is rapidly followed by modulation of bAPP synthesis in parallel to, or preceding, morphological changes of both microglia and astrocytes. The present study, which extensively characterized early changes in c-fos expression and bAPP-LI in glia following kainate-induced seizures, is a potentially useful animal model for the in vivo study of numerous facets of bAPP synthesis and the possible role of such processes in Alzheimer’s disease. r 1997 Academic Press

1 To whom correspondence should be addressed. Fax: 1972-26536075.

Senile plaques (31) and neurofibrillary tangles (62) are the two principal histopathological features of Alzheimer’s disease (AD). Senile plaques consist of an extracellular deposit of amyloid, the plaque core, surrounded by dystrophic neurites. In many cases of AD, amyloid deposits are also observed in the cerebral vasculature. From both the cerebral vasculature and senile plaques amyloid was isolated, sequenced, and mapped to the long arm of chromosome 21 (58, 61). This peptide (b/A4) varies in length from 39 to 43 amino acids. The b/A4 peptide was found to be a fragment of a larger protein, the amyloid precursor protein (bAPP). Although the neurotoxic mechanism of b/A4 remains to be resolved (52, 67, 69, 70), the aggregation of the b/A4 peptide fragments to filamentous deposits seems to be essential in the development of senile plaques (8, 11, 32, 37). A major obstacle to elucidating and treating AD has been the lack of an animal model. Recently, the production of transgenic mice that express high levels of human mutant bAPP (with valine at residue 717 substituted by phenylalanine) and which progressively develop many of the pathological hallmarks of Alzheimer’s disease, including numerous extracellular thioflavin S-positive b/A4 deposits, neuritic plaques, synaptic loss, astrocytosis, and microgliosis, has been reported (19). Although this model provides additional proof for the importance of bAPP expression and b/A4 deposition in AD pathology and also suggests the means for evaluating whether compounds that lower b/A4 production can produce useful effects in an animal model, the model is limited by the requirement that the development of the AD phenotype in these mice is conditional on the presence of a structural mutation in the bAPP gene (APP717V F). The majority of human AD patients do not exhibit such structural gene mutations (4), suggesting that the relevancy of the transgenic mouse model to nonfamilial AD remains to be demonstrated. A number of experimental paradigms in animals as well as some clinical conditions in man, including head trauma (39, 44), epilepsy (53), excitatory amino acids (54, 55, 57, 63), ischemia (3, 29), needle-stab (46), heat

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shock (1), interleukin 1 (10), nerve growth factor (41, 56), and axotomy (51), have been shown to modulate brain bAPP synthesis. We chose kainate-induced seizures (KIS) to study the anatomical, morphological, and molecular mechanisms underlying brain bAPP synthesis for the following reasons: (i) KIS involve activation of specific and well-characterized brain regions (5, 66) which are interconnected by major neuronal pathways; (ii) in both KIS-induced injury and human AD, similar brain regions are affected, particularly limbic structures including the piriform and entorhinal cortex, hippocampus, and amygdala (6, 33, 50, 57); (iii) KIS are characterized by changes in brain levels of bAPP mRNA isoforms similar to those observed in AD (22, 57, 68); (iv) kainate receptor binding has been reported by some investigators to be significantly increased in AD brains, particularly the hippocampus (20); (v) the KIS paradigm allows us to investigate the mechanisms underlying transsynaptic amyloid pathology in an animal model since KIS-induced bAPP upregulation is characterized by spread of damage from a ‘‘trigger zone’’ by activated transsynaptic pathways to target nuclei (36, 66); and finally, (vi) the KIS model also offers the opportunity in an animal paradigm to test drugs and other agents that may modulate bAPP synthesis. In the current study we employed immunohistochemical techniques to characterize and identify neuronal and glial cells in specific brain areas that modulate bAPP synthesis following KIS. In addition, antibodies directed against the FOS protein, which is generated by activation of the immediate early gene c-fos and is temporally associated with ongoing seizure activity during the first 24 h after kainate injection (34, 49), were used to identify KIS-activated transneuronal pathways. It was therefore possible to correlate the appearance of activated neuronal pathways identified by FOS immunoreactivity and bAPP immunoreactivity in alternate sections. In the past decade accumulating evidence demonstrates the remarkable role played by microglia and astrocytes in the brain (23, 30, 43, 59, 60). Astrocytes not only appear to assist neuronal function by providing growth factors including nerve growth factor, glialderived growth factor, and cytokines such as interleukins to surrounding nerve cells but they also participate in signal transmission by terminating the action of neurotransmitters and ions at the synaptic cleft. Of particular interest in the context of the KIS model is the removal by astrocytes of the excitatory amino acid, glutamate. Astrocytes actively take up glutamate, convert it to glutamine, and participate in its recycling back to the neuronal neurotransmitter pool. However, there is a possible negative aspect to the role of astrocytes in this recycling since glutamate may also weaken glial cells and disrupt their ability to supply

neurons with glutamine, thereby interrupting excitatory amino acid transmission (64). A second important glial cell type is microglia (23, 43, 59, 60), which are the first line of immune defence in the brain and function in a capacity similar to macrophages in the periphery. Not only do microglia assist in repairing and removing damaged neurons but there is also a dubious side to their activities since as part of their phagocytic and immunological function microglia generate free radicals, proteases, and cytokines that may aggravate destructive inflammatory processes and further amplify neurodegeneration. These considerations prompted us in the current study to employ immunohistochemical procedures using specific antibodies to characterize morphological changes in neuronal and glial cells following KIS (33) in both young and adult rats. METHODS AND MATERIALS

Injection Procedure Male albino Sabra rats (Sprague–Dawley origin) were housed in plastic cages and had ad lib. access to food and water. Kainic acid was dissolved in 0.1 M phosphate-buffered saline, pH 7.4, and was injected subcutaneously in the dorsal neck region. Behavior was monitored for 6 h and only rats that showed recurrent seizures from the second hour through the sixth hour were included in the study. Seizures Two hours following kainate injection all rats displayed characteristic behavioral manifestations including staring and wet dog shakes followed by repeated episodes of recurrent seizures. Such seizures included clonic forepaw movements that spread to elevation of the forequarters off the ground. Between these episodes, rats were either still and alert or made short lunges forward. The behavioral patterns observed in the current study are in agreement with earlier reports that described kainate-induced seizures (5, 33, 36). Preliminary experiments showed a differential sensitivity of young and old rats to seizure induction following a single dose of kainic acid. In order to compare the effect of KIS in young and adult animals, the dose of kainic acid was adjusted so that a similar course and intensity of seizures was observed in both age groups. Intensity and duration of behavioral seizures has been shown to correlate well with damage to brain tissue (33). Doses were therefore adjusted so that both age groups developed behavioral seizures within 1–2 h and subsequently displayed generalized seizures for 4 h. Six young rats, 8–9 weeks of age, received kainate 15 mg/kg. Five adult rats, over 1 year of age, received kainate 10 mg/kg.

bAPP IN RAT CORTEX AFTER KAINATE-INDUCED SEIZURES

Sacrifice and Tissue Preparation At 6 h post-kainate injection, rats were deeply anesthetized by intraperitoneal injection of nembutal. Brains were fixed by transcardial perfusion of ice-cold solutions: 50 ml of 0.02 M PBS, pH 7.4, followed by 200 ml of 4% paraformaldehyde in 0.1 M PBS, pH 7.4, and containing 4% sucrose. After perfusion, brain was immersed in the same fixative for another hour at 4°C. Brain blocks were immersed in 10% sucrose in 0.1 M PBS, pH 7.4, at 4°C and kept overnight. Sections for immunohistochemistry were 30 µm thick and were collected in PBS, transferred to a cryopreservation buffer, and kept at 220°C until immunohistochemical processing. The cryopreservation buffer contained 40% ethylene glycol and 1% polyvinylpyrrolidone in 0.1 M potassium acetate, pH 6.5. Sections for general cellular staining with cresyl violet were 15 µm thick and were collected by thaw-mounting onto gelatinized slides. Antibodies Two monoclonal antibodies directed against bAPP (clone 22C11, Boehringer Mannheim; and clone LN27, Chemicon) were used at a dilution of 1:500 for immunohistochemical identification of bAPP. These antibodies have been reported to stain astrocytes, microglia, and neurons in many studies (2, 65). No differences were observed between these two antibodies. A monoclonal antibody directed against the FOS protein (14) was used (Biomakor, Israel) at a dilution of 1:25,000. Microglia were identified using a monoclonal antibody (clone OX42, Cedarlane, Canada) directed against rat complement receptor 3 (CR3) at a dilution of 1:500. Astrocytes were identified using a monoclonal antibody directed against GFAP (clone G-A-S, Sigma Israel) at a dilution of 1:1000.

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cortex. A second set of drawings covers the dorsolateral cortex including retrosplenial cortex, somatosensory cortex, and parietal cortex. Camera lucida drawings were obtained from a single section representative of five sections examined for each rat in each of the two age groups. RESULTS Kainic Acid-Induced Seizures

As schematically illustrated in Fig. 1, previous studies (34, 49) have shown that KIS induce FOS-like immunoreactivity (-LI) in limbic structures including the amygdala (A) and hippocampus (H) and in cortical regions including the piriform (P) and temporal cortex (TC). Although FOS-LI is also typically induced in the mediodorsal thalamus (T) and hippocampus (H), the current investigation was limited to cortical and limbic regions. The Distribution of FOS-LI in Rats Displaying Seizures FOS-LI cells were detected in precisely circumscribed brain regions (Figs. 2 and 3) and differences were observed between young and adult animals (summarized in Table 1). In both young and adult rats FOS-LI cells were detected in the piriform cortex (Figs. 2B and 2C), whereas no FOS-LI is detected in the control section (Fig. 2B). In young rats FOS-LI is distributed throughout the limbic cortex (Fig. 2B) but in adult animals FOS-LI is restricted to the upper layer

Immunohistochemistry Immunohistochemical staining was performed following the standard biotin–avidin–peroxidase procedure (Elite Vecta Stain, California). However, the biotinylated secondary antibody (goat anti-mouse) was mouse Fab specific and preadsorbed on human IgG and on rat serum proteins (Sigma B0529). The objective was to avoid artifactual results related to species crossreactivity or Fc phenomena associated with microglial activation. In control experiments (not shown) we verified that if anti-APP antibodies were omitted no staining was obtained. Camera Lucida Illustrations Camera lucida drawings were prepared to document the anatomical distribution of cells stained with a given antibody. One set of drawings covers the piriform cortex, amygdala, endopiriform nucleus, and temporal

FIG. 1. Cross section of the rat brain (47). Each set of data is presented by two camera lucida drawings, one covering the ventral structures: TC, temporal cortex; E, endopiriform nucleus; A, amygdala; P, piriform cortex; and RF, rhinal fissure. The other camera lucida drawing covers RSC, retrosplenial cortex; SM, somatosensory and motor cortex; PC, parietal cortex. Other brain regions are HYP, hypothalamus; H, hippocampus; and T, thalamus.

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FIG. 2. Camera lucida, FOS-LI cells in the limbic cortex. (A) In a rat not injected with kainic acid FOS-LI cells were not detected. (B) In a young rat, seizures induced FOS-LI throughout the limbic cortex. (C) In an adult rat, seizures induced FOS-LI in the upper layer of the piriform cortex but not in the deeper layers or in the amygdala and endopiriform cortex.

II of the piriform cortex (Fig. 2C) and no immunoreactivity was observed in the deeper layers (V and VI) or in the amygdala and endopiriform cortex. In young rats mild and diffuse induction of FOS-LI was seen in the dorsolateral cortex (Fig. 3B), whereas in adult rats, intense FOS-LI was induced in the dorsolateral cortex, including all layers in the retrosplenial and somatosensory cortex and in layers 2–3 of the parietal cortex (Fig. 3C). No FOS-LI was observed in control sections (Fig. 3A). In summary, in adult rats kainate-induced FOS-LI spread outside the domains of the limbic structures (piriform and entorhinal cortex and amygdala) into cortical association areas (parietal and temporal cortex). In young rats, FOS-LI remained confined to the limbic-olfactory regions. The Distribution of bAPP-LI Control animals. In the absence of seizures some pyramidal neurons (Figs. 4, 5A, 5C, 6A, and 6C) and glial cells (Figs. 5A, 5C, 6A, and 6C) were bAPP-LI positive in the deeper cortical layers. In the amygdala and piriform cortex bAPP-LI glia were more prevalent than bAPP-LI neurons (Fig. 5). Seizures. The number of bAPP-LI neurons decreased in rats of both age groups (Figs. 5B and 5D vs 5A and 5C and Figs. 6B and 6D vs 6A and 6C), whereas the number of bAPP-LI glia increased dramatically in adults (Figs. 5D vs 5C and 6D vs 6C). At this time period, cresyl violet failed to show neuronal fallout. These changes were observed in both limbic and tempo-

ral cortex (Fig. 5) and in the somatosensory-motor and retrosplenial cortex (Fig. 6). In young rats fewer bAPPLI glia were observed (Figs. 5A, 5B, 6A, and 6B) both before and after seizures in comparison to the adult animals. In addition, differences were observed in the anatomical distribution of bAPP-LI glia following seizures. In young animals an increased number of bAPPLI glia were observed in the endopiriform nucleus and deep temporal cortex (Fig. 5B), whereas in adult animals, the number of bAPP-LI glia were increased in the piriform and temporal cortex (Figs. 5C and 5D) but not in the endopiriform nucleus. In summary, there was a decrease in bAPP-LIpositive neurons in both young and adult animals in animals with seizures. However, there was no evidence at this time period indicating neuronal fallout. There was a marked increase in bAPP-LI glial cells following seizures, which in adult animals was also observed outside the limbic brain regions. Distribution of Complement Receptor 3 (CR3)-Immunoreactive (ir) Microglia At least three microglial morphological types, reflecting distinct functional activities, are observed in brain (70). Microglia are generally found in their resting, highly ramified state. However, following brain injury and in the presence of dystrophic neurons, they retract their branches and migrate to the site of injury and assume a new morphological appearance. The precise shape usually depends on the architecture of the brain

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Seizures. Although type 1 microglia were also present in animals with recurrent seizures, there was a marked increase in microglia that showed more intensive staining of the cell body and the dendrites (Fig. 7A, type 2). Some microglial cells also appeared to undergo a more profound transformation which was characterized by a diminished or totally absent dendritic tree (Fig. 7A, type 3). In young rats, type 2 microglia were present in the upper layers of the piriform and temporal cortex (Fig. 8B), albeit in adult rats few type 2 microglia appeared in these brain regions (Fig. 8D). In young rats, type 3 microglia appeared in the amygdala and endopiriform nucleus (Fig. 8B), whereas in adult rats, type 3 microglia appeared not only in all of the above regions but were also observed in the piriform cortex (Fig. 8D). In young rats with seizures, the dorsolateral cortex appeared unchanged in terms of microglial morphology (Fig. 9B), whereas in adult rats there was extensive alteration in the distribution of morphological types in this region (Fig. 9D). In adult rats, type 2 microglia appeared in the upper layers of the retrosplenial and somatosensory cortical regions (Fig. 9D) and type 3 microglia were observed in the deeper layers of these regions (Fig. 9D). In adult rats, a preponderance of type 3 microglia were observed in the parietal and layers 2–4 of the temporal cortex (Fig. 9D). In summary, two types of effect were detected during seizures: (1) a hypertrophic effect, which was characterized by the appearance of type 2 microglia, and (2) a transforming effect, which was distinguished by a transition from type 1 to type 3 microglia. The hypertrophic effect was associated with specific brain regions and was more common in the young than in the adult rats. The transforming effect was more common in adult rats. FIG. 3. Camera lucida, FOS-LI cells in the parietal, somatosensory, and retrosplenial cortex. (A) In a rat not injected with kainic acid, FOS-LI cells were not detected. (B) In a young rat, seizures induced a few FOS-LI cells, mainly in the somatosensory cortex. (C) In an adult rat, seizures induced FOS-LI throughout the dorsolateral cortex and retrosplenial cortex.

region in which the microglia find themselves. Three states of microglia are generally observed: (i) state 1, or the resting state, characterized by ramified cells, (ii) state 2, or newly activated, which assume various morphological shapes including bushy, rodlike, and perineuronal, and (iii) state 3, or phagocytic, in which microglia attempt to degrade dead matter. The following microglial types were observed in the current study. Control rats. The common form of microglia, seen in control animals, was characterized by moderate staining of the cell body and dendritic tree (Fig. 7A, type 1). In both adult and young control animals, type 1 microglia were regularly scattered throughout the cortex (Figs. 8A, 8C, 9A, and 9C).

Distribution of Glial Fibrillary Acidic Protein (GFAP)-LI Astrocytes Control animals. Typical astrocyte morphology was observed (Fig. 7, type 1) and type 1 astrocytes were regularly spaced throughout the cortex (Figs. 8A, 8C, 9A, and 9C) in both young and adult animals. Seizures. Astrocytes were observed with distorted cell bodies and dendrites. The cell body was either swollen or shrunken and the dendrites either diminished in size or absent (Fig. 7, type 3). In both young and adult rats, type 3 astrocytes appeared in the vicinity of the basolateral amygdala and in the endopiriform nucleus (Figs. 10B and 10D). In adult rats type 3 astrocytes also appeared in the deep layers of the piriform and temporal cortices (Fig. 11D). Type 1 and type 2 astrocytes were preserved in the upper layers of the piriform cortex (Fig. 10D). A different pattern of astrocytic morphology was observed between the dorsolateral cortex of young and adult rats (Figs. 11B and 11D). In young rats, astrocytic morphology was not altered during seizures except for

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TABLE 1 Summary of Changes Following Kainate-Induced Seizures Age

Brain region

Young

Amygdala Endopiriform nucleus Piriform cortex Temporal cortex Parietal cortex Somatosensory cortex Retrosplenial cortex Amygdala Endopiriform nucleus Piriform cortex Temporal cortex Parietal cortex Somatosensory cortex Retrosplenial cortex

Adult

FOS-LI cells 111 111 111 (All layers) 1 (Layers 5–6) 1 (Layers 2–6) 1 (Layers 2–6) 1 (Layers 2–6) 11 (Layers 2–3) 111 (Layers 2–3, 5–6) 111 (Layers 2–3) 111 (Layers 2–6) 111 (Layers 2–6)

APP-LI glia

CR3-LI microglia

GFAP-LI astrocytes

1 111 1 (Layers 2–3)

111 111 111

111 111 11

1 (Layers 5–6) 1 (Layers 5–6) 11 (Layers 1–2) 1 1 11 (Layers 2–3) 111 (Layers 2–6) 111 (Layers 2–5) 111 (Layers 2–4) 111 (Layers 2–5)

111 111 111 111 (Layers 2–4) 111 (Layers 2–3) 111 (Layers 2–4) 111 (Layers 2–5)

11 11 11 111 (Layers 3–4) 111 (Layers 3–4) 111 (Layers 3–4) 11 (Layers 2–4)

Note. Degree of change is represented by the following symbols: low 1, moderate 11, high 111. Note that with regard to FOS-LI and bAPP-LI, change means that more cells expressing the antigen were detected, whereas with regard to CR3-LI and GFAP-LI, change means that more cells with morphological alterations were detected. Furthermore, in young rats, morphological alterations are mostly from type 1 to type 2, whereas in adults morphological changes also include changes to type 3. In parentheses are specified the cortical layers in which change was detected.

some changes in layer 1 (Fig. 11B), whereas in adult rats there were significant increases in type 3 astrocytes in layers 2–4 of the parietal cortex (Fig. 11D). In summary, the morphological changes in astrocytes that were observed following KIS were not within the normal range of morphological variation. Reactive astrocytes generally maintain a full dendritic tree, whereas following KIS we observed dendrites that were either distorted or entirely lost. This pattern of distorted dendrites was accompanied by either shrunken or swollen cell bodies. In contrast, the KIS-induced changes in microglia morphology that we observed were similar to those previously reported following treatments that induce microglia activation. Relationship between Morphological Markers and Their Anatomical Distribution Following KIS

FOS and bAPP A coincidence of bAPP-LI glial cells and FOS-LI cells was consistently observed in several brain areas. This coincidence was particularly striking in layers 2–3 of the parietal cortex from adult rats. In this region FOS-LI cells (Fig. 3C) are quite prominent and their presence coincided with an abundant number of bAPPLI glial cells (Fig. 6D). In contrast, the number of bAPP-LI neurons was markedly diminished. Conversely, in those regions showing only weak FOS-LI (Fig. 3C) only a few APP-LI glial cells were observed (Fig. 6D) and bAPP-LI neurons were preserved. However, in young rats a dissociation was occasionally noted in some regions between FOS-LI and bAPPLI. For example, while there was extensive FOS-LI in the piriform cortex (Fig. 2), bAPP-LI glia were not

FIG. 4. bAPP immunohistochemistry. (A) Control rat, pyramidal neurons in layer 4 of the cerebral cortex. (B) Rat with seizures, glial cells in layers 2–3 of the cerebral cortex. Calibration bar, 50 µm.

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FIG. 5. Camera lucida. Effect of seizures on the distribution of bAPP-LI cells in ‘‘limbic’’ and temporal cortex. Note the presence of bAPP-LI glia in the amygdala and deep piriform cortex in both young and adult controls. Note that in young rats induction is restricted to the endopiriform nucleus, whereas in the adult induction includes the superficial layers of the piriform and all layers of temporal cortex. Triangles, neurons; dots, glia.

observed in this region (Fig. 5B). On the other hand, FOS-LI was prominent in the endopiriform nucleus and amygdala (Fig. 2B) and coincided with extensive bAPP-LI glia in the basolateral amygdala and in a restricted region within the endopiriform nucleus (Fig. 5B). The dissociation of bAPP-LI and FOS-LI was also seen in the absence of seizures: bAPP-LI glia were observed within the amygdala (Figs. 5A and 6A) in the absence of FOS-LI in this structure (Fig. 2A). FOS and Glial Markers Microglia. Following seizures, the appearance of type 3 microglia generally coincided with intensive FOS-LI staining. This was particularly evident in the dorsolateral cortex: In layers 2–3 of the parietal cortex, intensive FOS-LI was coincidental with transformation of microglia from type 1 to type 3 in the same layers (Fig. 9D). Conversely, in deeper layers weak FOS-LI was observed and most microglia were type 1 (Fig. 9D). Similarly, in young animals with seizures, almost no FOS-LI was detected in the dorsolateral cortex (Fig. 3B) and no morphological transformation (type 1 to type 3) was observed in microglia in this brain region (Fig. 9B). Exceptions to these observations were occasionally encountered. For example, the entire complex of amygdala, endopiriform nucleus and piriform cortex showed intense FOS-LI (Fig. 2B) in young animals, whereas microglial transformation to type 3 was restricted to the amygdala and endopiriform cortex. Such

a dissociation between FOS-LI and microglial activation was also occasionally observed in adult rats following seizures. For example, type 3 microglia were observed in the amygdala and endopiriform cortex (Fig. 3D) in the absence of intensive FOS-LI (Fig. 2C). In summary, KIS-induced morphological changes in microglia are usually, but not invariably, associated with intensive FOS-LI. Overall, the coincidence between FOS-LI and microglial transformations is more pronounced in adult animals. Astrocytes. In adult rats with seizures some brain regions contained patches of astrocytes with altered morphology (Fig. 11D) which coincided with intense FOS-LI (Fig. 3C). In young rats these changes were restricted to the endopiriform nucleus (Figs. 2B and 10B). For example, in the dorsolateral cortex of young rats with seizures, no changes in astrocytic morphology were observed (Fig. 11B) and the absence of transformed astrocytes coincided with weak FOS-LI in this region (Fig. 3B). In summary, although an overall correlation was observed between changes in astrocyte morphology and FOS-LI in rats with seizures this coupling appeared to be strongest in adult rats. bAPP and Morphological Alterations in Glial Cells

Microglia Overall, there was a coincidence of type 3 microglia and bAPP-LI glia.

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FIG. 6. Camera lucida. Effect of seizures on the distribution of bAPP-LI in somatosensory-motor and retrosplenial cortex. Note the presence of bAPP-LI neurons in both young and adult controls and that this staining is attenuated by seizures in the same cortical subregions. Neuronal staining is spared in both retrosplenial cortex and deep parietal cortex. Note that adult control rats had more bAPP-LI glia than young control rats. In the adult seizures induced extensive staining of bAPP-LI glia. Triangles, neurons; dots, glia.

For example, in adult rats with seizures, layers 2–3 of the parietal cortex contained both type 3 microglia (Fig. 9D) and bAPP-LI microglia (Fig. 6D). Conversely, in deeper layers of parietal cortex, type 1 microglia (Fig. 9D) and bAPP-LI neurons (Fig. 6D) were preserved. Similarly, in young rats with seizures, microglia were not transformed in the dorsolateral cortex (Fig. 9B) and few bAPP-LI glia were observed. Peculiarly, as observed in adult animals (Fig. 6B) the number of bAPPLI neurons was diminished. In young animals with seizures (Fig. 8B) type 3 microglia were abundant in the endopiriform nucleus and coincided with an increased number of bAPP-LI glia (Fig. 5B). However, in an adjacent region the piriform cortex (Fig. 8B), there was a dissociation between the number of type 2 microglia and bAPP-LI glia (Fig. 5B). Similarly, in adult rats with seizures, the entire complex of the amygdala, endopiriform nucleus, and piriform and temporal cortex contained type 3 microglia (Fig. 8D), whereas bAPP-LI glia were observed only in discrete subregions (Fig. 5D). In summary, following KIS a more extensive coincidence was observed between the appearance of bAPPLI microglia and microglial transformations (type 1 to type 3) in adult compared to young rats. The correlation

in young rats between bAPP-LI and microglia transformation was more restricted and was limited to specific brain regions. Astrocytes The appearance of damaged astrocytes generally coincided with bAPP-LI glia. For example, in adult rats with seizures transformed astrocytes were seen in layer 4 of the parietal cortex (Fig. 11D) and coincided with the appearance of bAPP-LI glia (Fig. 6D). An occasional exception was observed. For example, in the upper layers of this region the presence of bAPP-LI glia (Fig. 6D) did not coincide with the appearance of abnormal astrocytic morphology. DISCUSSION

We have used a model of kainate-induced seizures to investigate the cellular mechanisms involved in accumulation of bAPP in specific brain regions. In addition to identifying bAPP-LI, we also monitored seizureinduced transsynaptic activation by immunohistochemical demonstration of FOS-LI. Finally, we identified morphological changes in both astrocytes and microglia

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FIG. 7. Immunohistochemical staining with marker antibodies for glial cells. (A) Microglia stained by anti-CR3 (OX42). Type 1 is the normal ramified state in which processes are weakly stained and the cell body sometimes not stained at all. Type 2 is also in the ramified state but both processes and cell body are intensely stained. Type 3 has a round or ovoid cell body with a short stub or two coming out of it and is similar to the description of active microglia. (B) Astrocytes stained by anti glial fibrillary acidic protein (GFAP). Type 1 is the normal state, Type 2 is in the active state, as the cell body is stained in addition to the processes. Type 3 is either a residual structure consisting of a ‘‘balooned’’ or swollen astrocyte with one or two processes coming out of it or a small shrunken cell body with processes that are tortuous instead of the characteristic smooth processes of healthy astrocytes. Note the accumulation of astrocytes around a blood vessel (V). This was not seen with microglia. Calibration bar, 50 µm.

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FIG. 8. Camera lucida. Effect of seizures on the distribution of CR3-LI cells (microglia) in limbic and temporal cortex. In both young and adult control rats, type 1 microglia were regularly scattered throughout the cortex. In the young, type 2 cells were induced in the superficial layers of the piriform and temporal cortices but only in the temporal cortex in adult. Type 3 microglia were induced in the amygdala and endopiriform cortex in the young but in the entire limbic and temporal cortex of adults. (x) Type 1; (*) type 2; (W) type 3.

induced by KIS and correlated such changes with bAPP-LI and FOS-LI at the regional and cellular level. Seizures are known to induce the activation of the immediate early gene c-fos and the anatomical profile of neuronal activation following seizures, as revealed by FOS immunohistochemistry, has been well characterized (34, 66). Our results are consistent with previous reports (34, 49) which demonstrate a specific pattern of FOS-LI induced by kainate injection. In the current investigation we have extended these studies and compared the seizure pattern in young and adult rats. In older animals FOS-LI spreads out from limbic cortical regions (piriform, entorhinal) to other cortical regions (parietal, somatosensory cortices). In the present study changes in morphology and histoimmunochemical markers following KIS were assessed at one time period, 6 h after kainate administration. La Salle (34) and subsequently Popovici et al. (49) described KIS-induced c-fos oncogene expression and showed that the time course of FOS-LI appearance in various brain regions differed according to the anatomical site. It is not unlikely that the KIS-induced synthesis of bAPP-LI follows a similar pattern. The current results should therefore be cautiously interpreted since

KIS-induced changes in FOS-LI occur very rapidly in discrete brain regions and these dynamic changes are only partially reflected by assessment at a single time point. In particular, failure to observe association between FOS-LI and bAPP-LI in some regions may reflect regional, kinetic, and age-associated differences in the rate of synthesis between these two proteins and not a true dissociation between FOS and bAPP induction. In our model, KIS were associated with decrease in neuronal bAPP-LI staining in both young and adult rats, whereas glial bAPP-LI markedly increased. The loss of neuronal bAPP-LI cannot be simply attributed to neuronal fallout since at this time interval cresyl violet staining fails to reveal any neuronal cell loss. However, our observations are consistent with a previous report showing that APP695 mRNA, a neuronalspecific isoform of bAPP, is decreased following KIS (57, 68). Further studies are required to clarify the reasons for the FOS-induced decrease in neuronal bAPP-LI. The increase in bAPP-LI glia was far more extensive in adult than in young rats and the anatomical distribution of bAPP-LI glia was grossly correlated with FOSLI. Our findings lend support to the concept that the spread of bAPP-LI follows KIS-activated transsynaptic

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FIG. 9. Camera lucida. Effect of seizures on the distribution of CR3-LI cells (microglia) in somatosensory-motor and retrosplenial cortex. In both young and adult control rats type 1 microglia were regularly scattered throughout the cortex. In young rats with seizures this was essentially the same except for some type 2 microglia in the retrosplenial cortex. In adult rats with seizures, type 3 microglia appeared in layers 2–3 in the parietal and somatosensory cortex, but type 2 microglia were also seen in layer 1 of the retrosplenial and somatosensory cortex. (x) Type 1; (*) type 2; (W) type 3.

pathways. Hardy (25, 26) recently hypothesized that in AD (1) bAPP is initially deposited in the limbic areas for unknown reasons, (2) deposition of bAPP induces stress in the surrounding neuropil, (3) the stress response increases the synthesis of bAPP and other proteins (apolipoprotein E, anti-chymotrypsin, interleukins) in the plaque area, which further increases bAPP accumulation, and (4) axonal transport of bAPP leads to increased bAPP accumulation at the terminals of damaged neurons, thus amplifying the damage, and leads to the propagation of the damage along fixed neuronal pathways. This so-called ‘‘amyloid cascade hypothesis’’ suggests an explanation at the molecular level for the anatomical pattern of plaque distribution described in AD by Pearson et al. (48) and by Lewis et al. (35), who demonstrated that there is a precise neuroanatomy of plaque distribution and that the disease process appears to spread along connecting fibers which link affected regions. One of our considerations in choosing the KIS model to modulate bAPP synthesis is the similarity between the distinct pathological distribution of plaques in AD and the spread of kainate-induced damage in rat brain. The differences observed in the present study between young and adult rats provides some support for the

conjecture that with increasing age there is augmented vulnerability to upregulation and accumulation of bAPP. Our results strengthen previous findings that older rat brains are more vulnerable to kainate-induced damage (71). In adult brain it has also been shown that microglia are the responding cell type to lesion, whereas in immature rats macrophages are the predominant responding cell class (40). Some of the differences observed in the present study, which demonstrate an increased microglial involvement in response to KIS in the adult animals, are consistent with these observations. For reasons only partially understood, specific brain regions respond to KIS with intensive synthesis of glial cell bAPP. It is not unlikely that insult to the human brain due to a variety of causes, including head trauma (39, 44), convulsions (53), ischemia (3, 29), lesions (54–56, 63), and environmental agents, induce focal points of glial cell amyloidosis which represent the first stages of plaque formation. Such environmental insults may be synergistically coupled with genetic vulnerability conferred by polymorphisms such as the apolipoprotein E4 genotype (12, 13). It is interesting to note that reactive glia are the apparent source for apolipoprotein E4 in brain (37). Further studies employing the current KIS rat model of bAPP upregulation

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FIG. 10. Camera lucida. Effect of seizures on the distribution of GFAP-LI cells (astrocytes) in limbic and temporal cortex. In both young and adult control rats, type 1 astrocytes were regularly scattered throughout cortex and there were type 2 astrocytes in the superficial layers of the piriform cortex in young rats. In young rats with seizures, damaged astrocytes (type 3) appeared in the amygdala and endopiriform nucleus and some in the deep piriform cortex. In adult rats, type 3 astrocytes appeared in the same regions but also in layers 2–5 of the temporal cortex. (x) Type 1; (*) type 2; (W) type 3.

may shed light on some aspects of the mechanisms governing bAPP accumulation in human brain. The immunohistochemical methodology employed in the present study does not allow us to unequivocally identify FOS, bAPP, and cell type markers OX42 (microglia) and GFAP (astrocytes) on individual cells in the same section. However, using alternately stained sections and by careful attention to morphological characteristics, we feel confident in our observation that there is an overall upregulation of bAPP-LI in activated microglia and astrocytes. It would appear that the microglia and astrocytes are synthesizing bAPP. The possibility cannot be excluded, however, that microglia are also ingesting amyloid by phagocytosis. We consider this possibility unlikely since we found little if any evidence for neuronal necrosis that could potentially provide an extracellular source of microglial amyloid. However, future in situ hybridization studies using mRNA probes to isoforms of bAPP (57, 68) will provide information both on the identity of cells generating bAPP in the KIS model and on the relative contribution of these isoforms (52). It is likely that the sequence of events following KIS is initially triggered by c-fos gene expression, which is rapidly followed by modulation of bAPP synthesis in parallel to, or preceding, transformation of both microg-

lia and astrocytes into their activated states. The rapid upregulation of bAPP-LI, which occurs in parallel with transformation of both glial cell types, suggests that bAPP plays a key role in these processes. One of the roles attributed to bAPP is cell adhesion (52), suggesting that activation of microglia and their migration to sites of brain injury may be conditional upon increased bAPP synthesis. Furthermore, it has been demonstrated that transcription of the bAPP gene is stimulated in vitro in human NT-2 and HeLa cells by various types of stress (16). Since seizures involve stress to both neurons and glia (33), it is relevant that in studies of global or focal ischemia in rats the expression of the stress-response proteins heat shock protein-70 and heme oxygenase 1 or 2 varies between neurons and glia depending on the brain region and with time after the trauma (21). Dalton et al. (15) report that the messenger RNAs of heat shock protein-70 and heme oxygenase 1 increase after KIS. Although there is no information on the distribution of these changes in neurons vs glia in the KIS model, future studies may reveal linkage between the type of stress-response protein activated and the class of cells overexpressing bAPP. The pattern of KIS-induced expression of glial cell bAPP-LI observed in the present study adds to the

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FIG. 11. Camera lucida. Effect of seizures on the distribution of GFAP-LI cells (astrocytes) in somatosensory-motor and retrosplenial cortex. In both young and adult control rats, type 1 astrocytes were regularly scattered throughout cortex. Only in adult rats with seizures did type 3 astrocytes appear in layers 2–4 of the parietal and somatosensory cortex. (x) Type 1; (*) type 2; (W) type 3.

accumulating evidence that astrocyte and microglia partially contribute to the initiation and development of AD plaques. In AD both types of glia were found closely associated with neuritic plaques (17, 28, 38). It is interesting to note that in man regional differences have also been observed in reactive gliosis and that reactive astrocytes are associated most strongly with senile plaques in the cortex and hippocampus (27). Only cortical and hippocampal astrocytes reacted intensively to immobilized bAP when challenged in in vitro experiments (27). Similarly, in the current study we observed that cortical regions are particularly sensitive to glial cell bAPP upregulation following KIS in adult rats. The role of microglia in the brain has become the focus of intense interest due to their role as resident brain macrophages and these cells have been demonstrated to function as phagocytes in reacting to brain injury (23, 30, 43, 59, 60). On the other hand, microglia have also been hypothesized to operate negatively in brain injury and may under some circumstances exacerbate inflammatory processes and induce additional neuronal damage. There is evidence that both of these processes are involved in AD plaque formation. In AD microglia become activated and aggregate around se-

nile plaque b-amyloid and neurofibrillary tangles. However, heavy accumulation of these pathological debris in postmortem indicates the failure, or at best partial success, of the removal. It is supposed that continued activation of microglia in these lesions elicits a persistent inflammatory response. At the molecular level numerous reports (3, 7, 9, 18, 22, 28, 29, 42, 43) demonstrate that microglia synthesize bAPP, which may directly contribute to amyloidosis and plaque formation. Moreover, this molecule and its derivatives may further induce amyloid and cytokine synthesis, resulting in a reinforcing feedback loop. Once the neurodegeneration cascade is initiated, microglial and astroglial cells may play major roles in directly and indirectly promoting self-sustaining neurodegeneration cycles. The rapid appearance of transformed microglia following KIS coincident with bAPP-LI indicates that these cellular elements, which are known to synthesize bAPP, are a possible locus for amyloidogenesis in the brain (35, 48). The KIS model for upregulating bAPP is a convenient system for evaluating the role of microglia and astrocytes in contributing to the amyloid load. It is interesting to note in the context of the present investigation that human astrocytes generate higher levels of

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bAPP than any other cell type examined (9). Although senile plaques are not normally observed in rodent brains, it is nevertheless of some interest to ascertain the duration of the microglial and astrocyte bAPP upregulation using the KIS model. We are aware of one previous report (54) which demonstrated higher levels of microglial bAPP-LI over 1 month after KIS. The duration of the KIS-induced upregulation of glial cell APP synthesis may be informative concerning the role of these cellular elements in amyloidosis in man. The KIS model offers the opportunity to examine some aspects of both the cellular and molecular consequences of long-term changes in regulation of amyloid synthesis. Although in the current report we examined immunohistological changes very shortly after kainate administration, experiments are now underway in our laboratory to examine the time course of amyloid upregulation employing both immunohistochemical and molecular methodologies. A direct role has been suggested for excitatory amino acid pathways in AD, adding an additional dimension to the relevance of the KIS model to the accumulation of APP in human brain (20). Anatomical and biochemical evidence suggests that there are both pre- and postsynaptic disruptions of excitatory amino acid (EAA) pathways in AD (24). Dysfunction of EAA pathways may also contribute to the clinical manifestations of AD such as memory loss since EAA have been postulated to be involved in memory activities. Furthermore, EAA might be involved in the pathogenesis of AD, by virtue of their neurotoxic properties. Circumstantial evidence raises the possibility that the EAA system may partially determine the anatomical distribution of pathology in AD. In this context it is interesting to note that forebrain cholinergic neurons are selectively vulnerable to AMPA/kainate receptor-mediated toxicity (50). These findings suggest that study of KIS-induced bAPP accumulation in rat brain could contribute to a more complete understanding of AD etiology in man.

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ACKNOWLEDGMENTS This work was supported in part by Grant 2917 from the Israeli Ministry of Science and the Arts in cooperation with the European Community and in part by the Joseph Levy Charitable Foundation (London), the Heinz and Anna Kroch Foundation (London), and the Shapiro–Grover–Leff–Simon families.

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