Transitory Disappearance of Microglia During the

GLIA 1252-61 (1994)

Transitory Disappearance of Microglia During the Regeneration of the Lizard Medial Cortex C. LOPEZ-GARCIA,' J. NACHER,' B. CASTELLANO: J.A. LUIS DE LA IGLESIA,l AND A. MOLOWNY' 'Neurobiolo ia, Biologia Celular, Facultad de Ciencias Biologicas, Universidad de Valencia, 46100 Burjasot, Hstologia, . Biologia Celular, Facultad de Medicina, Universidad Autonoma de Barcelona, and B 08193 Bellaterra, Spain

KEY WORDS

3-Acetylpyridine, Lesion, Hippocampus, Postnatal neurogenesis, Supraependymal cells

ABSTRACT In normal lizards, microglial cells populate the medial cortex (a zone homologous to the hippocampal fascia dentata), with a preferential distribution along the border between the granular cell layer and the plexiform layers. Intraperitoneal injection of the neurotoxin 3-acetylpyridine (3AP) induces a selective lesion in the medial cortex with a rapid degeneration of the granular layer and its zinc-enriched axonal projection. Within 6-8 weeks, the granular layer is, however, repopulated by a new set of neurons generated in the subjacent ependyma and the cell debris is removed. The aim of this study was to determine to what extent microglia were involved in the scavenging processes during the regeneration process. To this end we studied the brains of regenerating lizards at different times after 3AP lesion, visualising microglial cells by the nucleoside diphosphatase (NDPase) histochemical reaction. Surprisingly, we found that stained microglial cells disappeared 6-8 hours after 3AP injection and remained absent until 10-15 days after injection. One month postlesion an increased population of microglial cells was found scattered throughout all plexiform layers of the cortex. Thorough examination of semithin and ultrathin sections confirmed the absence of microglia in the medial cortex of recent lesioned animals but the presence of an exuberant population after 1month postlesion. In the tissue, phagocytotic scavenging was carried out by radial ependymocytes, not by microglia. o 1994 Wiley-Liss, Inc.

INTRODUCTION The role of microglial cells in the adult brain is not completely understood, but it is widely accepted that they belong to the monocyte/macrophagelineage (Boya et al., 1979; Ferrer and Sarmiento, 1980b). From an ontogenetical point of view, at the moment of nervous parenchyma vascularisation, these mesodermic cells enter the central nervous system as ameboid cells and dwell in the perivascular parenchymal areas (Fujimoto et al., 1987; Ling, 1981; Rio Hortega, 1932). Then, ameboid cells are transformed into ramified microglia,which after the appropriate stimulus may be activated and involved in removal of neuronal debris and other immunopathogenic activities (Ferrer and Sarmiento, 1980a; Finsen et al., 1993a,b; Stenwing, 1972; Streit et al., 0 1994 Wiley-Liss, Inc.

1988). In normal lizards, microglial cells populate the cerebral cortex, showing a preferential distribution in specific areas of the plexiform layers (Berbel et al., 1981; Castellano et al., 1991a). The lizard cerebral cortex can be divided into four main areas: the medial, the dorsomedial, the dorsal, and the lateral cortices. Most neuronal somata in these cortices are closely packed into conspicuous cell layers, sandwiched between adjacent inner and outer plexiform layers. This histological pattern closely resembles

Received January 7,1994; accepted May 4,1994. Address reprint requests Prof. C. Lopez-Garcia, Neurobiologia, Biologia Celular, Facultad de Ciencias Biologicas, Universidad de Valencia, 46100 Bujasot, Spain.

MICROGLIA AND LIZARD CORTEX REGENERATION

that of embryonic stages of cortical developmentin mammals and that of their adult hippocampus. The lizard medial cortex is homologous to the hippocampal fascia dentata of mammals on the grounds of comparable histochemistry, ontogeny, and position in the cortical circuitry (Lopez-Garcia et al., 1983, 1988; Molowny and Lopez- Garcia, 1978). It receives afferents from the lateral cortex (olfactory cortex) and the remaining cortical areas (dorsal and dorsomedial) in a highly laminated fashion (Martinez-Guijarroet al., 1990). On the other hand, the lizard medial cortex emits a zinc-rich axonal projection directed towards the dorsomedial and dorsal cortices (Lopez-Garcia et al., 19831, which has been considered homologous to the hippocampal mossy fibre system in mammals (Lopez-Garcia and Martinez-Guijarro, 1988; Martinez-Guijarro et al., 1991; Molowny and Lopez-Garcia, 1978). Lizards lesioned with the neurotoxin 3-acetylpyridine (3AP) show severe lesion of the medial cortex principal cell layer neurons. Surprisingly, this lesion is followed by intensive neurogenetic activity in its subjacent ependyma, giving rise to a new set of neurons that regenerate a new medial cortex with normal histological appearance (Font et al., 1991; Lopez-Garcia et al., 1990b). This regenerative process involves recruitment and differentiation of the newly produced neurons, as well as simultaneous removal of dead cells (Lopez-Garcia, 1993). Microglial cells could be expected to be involved in these processes. In order to clarify the role of microglia during cortical regeneration in lizards we studied the brain of 3AP-treated lizards, sacrificed at different times after 3AP injection. Microglial cells were labelled using the nucleoside diphosphatase (NDPase)histochemical reaction (Castellano et al., 1984; Sanyal and DeRuiter, 1985). MATERIALS AND METHODS Healthy adult specimens (4-5.5 cm snout-vent size) of the lizard Podarcis hispanica captured in the surroundings of Burjasot (Valencia),were used in this study. They were maintained in terraria, simulating their natural habitat conditions. The lizards were intraperitoneally injected with the antimetabolite neurotoxin 3AP (150 mg kg-' b/w) and sequentially sacrificed a t different times afterwards, i.e., 6 h, 8 h, and 12 h and 1 , 2 , 3 , 4, 5, 7, 10, 15, 30, and 180 days after 3AP administration. Non-injected lizards were used as controls. Under ether anaesthesia, the specimens were transcardially perfused with the fixative solution. Electron Microscopy For electron microscopy the specimens were perfused with a solution containing 2% glutaraldehyde, 2% paraformaldehyde in 0.1 M phosphate buffer (pH 7.2). Brains were removed and immersed for 4-6 h at 4°C in

53

the same fixative. Transverse sections (200 km thick) were obtained using a Vibratome. The slices were postfixed in 1%osmium tetroxide, 8%glucose in 0.1 M phosphate buffer (pH 7.2), for 2 h a t 4°C and then embedded in Durcupan. Toluidine blue-stained semithin sections were used to assess the extent of lesion in the brain. After re-embedding, ultrathin sections from selected areas were cut, stained with uranyl acetate and lead citrate, and finally observed in the electron microscope. NDPase Histochemical Demonstration Specimens were perfused with 4% paraformaldehyde, 5% sucrose in 0.1 M cacodylate buffer (pH 7.4). The brain was removed, immersed in the same fixative for 4-6 h at 4°C) and then transversely sectioned (200 pm thick) using a Vibratome. Histochemical detection of NDPase activity was performed to label microglial cells, as previously described (Castellano et al., 1991b). In short, slices were transferred to the incubating solution containing 25 mg of the sodium salt of inosine 5'-diphosphate (Sigma; cat. no. I 4375) dissolved in 7 ml of distilled water, followed by addition of 10 ml of 0.2 M Trizma-maleate buffer (Sigma) (pH 7.41, 5 ml of 0.5% MnCl,, and 3 ml of 1%Pb(N03)2,in that order. This solution was freshly prepared and filtered immediately before use. The slices were incubated for 20 min at 37°C and then rinsed in cacodylate buffer 0.1 M, pH 7.4. Immediate visualisation of the reaction product was obtained by immersion of the slices in 2% ammonium sulphide for 2 min. The precipitate was stabilised by washing the slices in distilled water, immersing them in 1%silver nitrate for 1min, and finally washing again in distilled water. The histochemical reaction was controlled by incubating some slices of non-injected animals in a solution without inosine 5'-diphosphate, which resulted in complete absence of label. For examination of microglia, some slices were postfixed in 1%osmium tetroxide just before treatment with ammonium sulphide. These slices were embedded in Durcupan and processed for electron microscopy as previously described. Quantitative Study The ratio of normal versus pyknotic nuclei (Fig. 2) was obtained from cell counts performed on semithin sections from animals sacrificed at different times after 3AP injection. The transverse semithin sections were chosen from a commissural level and three sampling zones systematically chosen from dorsal, medial and caudal-ventral parts of the medial cortex cell layer. The numbers of normal and pyknotic nuclei were counted by using an lOOX oil objective; the final sample size varied from 80 to 120 celldspecimen. The numbers of microglial and supraependymary cells in the medial cortex (Fig. 4) were also counted on semi-

54

LOPEZ-GARCIA ET AL.

Fig. 1.


thin sections of the lizard medial cortex. The sampling area was all the medial cortex (including all layers and ependymal lining of the ventricle) appearingin the transversal semithin section. The number of cells in this area was related to the size of the sampling area (about 0.45 mm2/section).

55

100

Normal

80 60

RESULTS Semithin Section and Electron Microscopic Study

40 20

By 1day after 3AP treatment, 6695% of nuclei in the medial cortex cell layer appeared pyknotic. Between 1 and 8 weeks after 3AP injection, the incidence of pyknotic nuclei decreased as the regeneration proceeded. Animals allowed to survive for long periods postlesion displayed almost normal histology of their cerebral cortex, in agreement with previous studies (Lopez-Garcia, 1993; Lopez-Garcia et al., 1990b; Lopez-Garcia et al., 1992a). Time Course of Morphological Changes Induced by 3Ap Lesion Twelve hours after 3AP injection, many neuronal somata of the medial cortex cell layer appeared damaged. The initial lesion was assessed by chromatin condensation and acute cytoplasm swelling. (Fig. 1A). One day later, these somata showed pyknotic nuclei and disorganised cytoplasm, indicative of cell death (Fig. 1B). From day 2 to day 8 postlesion, the medial cortex cell layer displayed the same morphology. Many pyknotic nuclei, residual debris, and normal somata coexisted in the medial cortex cell layer, but some fusiform immature neuronal somata with fusiform or polarised cytoplasm and a dispersed chromatin configuration inside their nuclei (Fig. lC,D) appeared between the dead somata. This morphology corresponds to that of the migratory immature neurons frequently seen in the inner plexiform layer of these animals (Garcia-Verdugoet al., 1986). From day 8 t o day 30, many immature fusiform-tospherical neuronal somata were progressively recruited in the medial cortex cell layer (Fig. 1D). During this period, the number of pyknotic nuclei in the medial cortex cell layer progressively decreased, while the number of newly immature plus normal somata increased (Fig. 2).

Fig. 1. The lizard medial cortex (MC) in toluidine blue stained semithin sections after different survival times following 3-acetylpyridine injection ( A 12 h., B: 1 day. C: 8 days. D: 18 days. E: 40 days. F: 40 days.) Observe initial swelling of medial cortex granule cells and nuclear pyknosis 1 day after injection. At 8-18 days, the medial cortex cell layer remains disorganised with dead cell somata with pyknotic nuclei (arrows in C) and migratory immature neurons with elongated nuclei (asterisks). After 40 days the medial cortex displays an almost normal histological appearancewith the exception of abundant microglial-like cells (dark cells in E) and supraependymal cells (arrows in F). cl, cell layer; ipl+, inner plexiform layer zinc positive; ipl-, inner plexiform layer zinc negative;opl, outer plexiform layer; ep, ependyma. Scale bars = 100 km in A, B, and E; 25 pm in F; 10 pm in C and D.

n "

0,5 1

4,5 6,5 8

10,5 15

18 21

34

35

40

90 180

Days after 3AP injection

Fig. 2. Histogram showing the relative frequence of pyknotic (bars) versus normal (lines) nuclei in the granule neurons of the medial cortex after 3-acetylpyridine (3AP) injection. Data are percentages observed in semithin sections.

There were abundant microglia-like cells in the medial cortex on day 40 after 3AP injection (Figs. l E , 3A). Thorough examination of both semithin and ultrathin sections showed that these cells were not present in the medial cortex before day 15 after 3AP injection. Supraependymal cells with a morphology similar to microglia cells (Figs. lF, 3B) were observed in the ventricle. The areal density of supraependymal cells and microglia-like cells of the medial cortex observed in semithin sections increased between postlesional days 15 and 40 (Fig. 4). NDPase Histochemistry In controls, NDPase histochemical detection specifically labelled microglial cells, supraependymal cells, and the luminal surface of endothelial cells in the paired blood vessels which are characteristic of the lizard cortex. Microglial cells were found scattered in the plexiform layers but frequently concentrated in the borders of the cell layers. This distribution agrees with that previously observed in this species (Castellano et al., 1991a). Microglial cells located in the inner plexiform layer displayed processes in close contact with the ependyma (Fig. 3C); moreover, occasional scattered cell profiles in the ependyma were labelled (Fig. 3D). Six hours after 3AP injection, the number of microglial cells in the medial cortex was reduced. Eight to 12 hours after the 3AP injection, stained microglia were virtually absent in the medial cortex. Only a few vertical fusiform microglial cells were observed close to the ependymal layer of the medial cortex. In the rest of the other cortical and telencephalic areas microglial cells with normal appearance were observed. From 1to 7 days after 3AP injection, no stained microglial cells were found in the medial cortex of the 3AP-treated lizards (Fig. 5B,D),although they were abundant in other telencephalic and diencephalic areas (Fig.

56

LOPEZ-GARCIA ET AL.

Fig. 3. Microglia and supraependymal cells in the lizard medial cortex. A Electron micrography aspect of microglia-like cell in the plexiform layer of a lizard that survived 15 days after 3AP injection. B: Electron micrography of supraependymal cell lining the medial cortex ependyma, 15 days after 3AP injection. C: NDPase stained microglial

cells in the inner plexiform layer of the medial cortex of a control lizard; the ependyma appears tangentially sectioned. D NDPase stained cell profiles in the ependyma. Scale bars = 10 pm in A and B; 100 pm in C; 50 pm in D.

5C,E). Fusiform microglial cells, frequently arranged in a radial fashion, were noticeable in specimens allowed to survive for 15 days after 3Ap injection (Fig. 5F). An increased number of microglial cells, even greater than that of control specimens, was observed in the medial cortex 1month after 3Ap injection (Fig. 5G,H). In these cases, the distribution of microglial cells was clearly different from that of the controls as the microglial cells populated all the plexiform layers, including the laminae that normally were free of them, i.e., the zinc-rich synaptic fields in the dorsal and dorsomedial cortices and the outer plexiform layers of the medial cortex.

DISCUSSION Lesion Mechanism of 3AP: Necrosis of the Medial Cortex Granule Cells The neurotoxin 3-acetylpyridine is selectively toxic for the principal neurons of the medial cortex, while it leaves the rest of cortical areas essentially unaffected (Lopez-Garcia et al., 1992a). Although the mechanism is not completely understood, it seems that this antimetabolite prevents ATP synthesis, thus being fatal to neurons with high activity during the drug administration. In this sense the medial cortex granule cells are likely targets, as they express C-fos immunoreactivity,


50

r"\

40

and increase the population of microglial cells. After a perforant path lesion, the increase in microglia numbers precedes astrocyte increase in the rat dentate gyrus (Fagan and Gage, 1990; Jensen et al., 1994). In contrast, 3AP lesion in lizards resulted in a different response: early activation of ependymocytes(Lopez-Garcia et al., 1992a) and loss of stained microglia from the lesioned area followed by an increase in microglia cell numbers in the overall cortex.

microglia

n

30

20

57

10

0Ctr. 0,5 1

Nature of the Fast Disappearance of Microglia 2

3

4

5

6

7

10

15

30

40

90

Days after 3AP injection Fig. 4. Histogram showing the areal density of microglia-like cells (microglia) and supraependymal cells (sup. cells) along the lesionregeneration process. Density values are number of cells per 2 x lo5 pm2,as seen in semithin sections. Superimposedin the line drawing is the hemisphere total number ( x 100) of ependymal cells (proliferating neuroblasts)labelled with either tritiated thymidine or 5-bromodeoxiuridine (unpublished data). (From Lopez-Garcia et al., 199213.)

a marker of neuronal activation (Sagar et al., 1988) in normal non-stimulated lizards (Blasco-Ibanez et al., 1992). As the loss of energy kills the neurons, they become swollen due to passive water intake after failure of the ATPase Na/K pump. The edema, the disrupted morphology of the cytoplasm displayed by afTected neuronal somata, and, finally, the delayed presence of debris all suggest a necrotic process in the lesioned medial cortex (Auer et al., 1985; Beiswanger et al., 1993). Debris Removal by Ependymoglia The cell debris was removed, rather inefficiently, by ependymocyticprocesses that engulfed and transformed it into lipofuscin bodies, which finally accumulated in the ependymocyte somata (Lopez-Garcia et al., 1992a). This was a long-lasting process that, in some circumstances, persisted for up to 9 months (unpublished observations). In amphibians, ectodennal cells such as ependymoglial-astrocytic cells have been found to be responsible for eliminating degenerated axons, while mesodermic cells play a minor role (Stensaas, 1983; Turner and Glaze, 1973; Turner and Singer, 1975; Zammit et al., 1993). 3AP Lesion Causes the Disappearance of Microglia One would expect that the lesion of the medial cortex, resulting in death of granule cells and edema, could be a signal for microglia activation. Drug or neurotoxin administration (OHearn et al., 19931, lesion (Akiyama and McGeer, 1989;Andersson et al., 1991; Ogawa et al., 1993),injury, and even seizures (Miyake and Kitamura, 1991; Shaw et al., 1990; Wilson et al., 1992) activate

The presence of histochemically labelled microglial cells in areas other than the medial cortex (in the same section) was evidence that something related to microglia was happening in the early lesioned medial cortex. A careful search for microglial-like cells in the semithin-ultrathin sections showed that the loss of NDPase histochemical staining was not due to a general loss of histochemical reactivity but to the absence of these cells in the early lesioned medial cortex. Although the exact mechanisms of disappearance, hypothetical microglia-apoptosis, and/or microglial emigration are unresolved, the absence of microglia in the lesioned medial cortex may contribute to the creation of a new microenvironment in which regeneration can take place. Microglial cells are invovled in the regulation of extracellular levels of adenosine, adenosine phosphate, and other nucleoside phosphates that have a role in the cell cycle (Fox and Kelley, 1978; Rathbone et al., 1992). Tritiated thymidine and 5-bromodeoxiuridine labelling techniques reveal a burst of neurogenesis ("reactive neurogenesis") in the ependyma of the medial cortex of lesioned lizards, from 2 to 7 days after 3AP lesion. Afterwards, the neurogenetical activity of the subjacent ependyma returns to a normal rate (Lopez-Garciaet al., 1992b). At this time, a new microglial population appeared in the medial cortex. It is tempting to suggest that microglial cells have a role in downregulating postnatal neurogenetical activity. The bone marrow-derived dendritic cell lineage is formed by a large number of antigen-presenting cells that stimulate primary and secondaryimmune responses (Brodsky and Guagliardi, 1991) with different names according their location (Moschella and Cropley, 1990; Sprecher and Becker, 1993) in different lymphoid and non-lymphoid tissues and organs, e.g., Langerhans cells in the skin and microglia in the nervous tissue (Lowe et al., 1989). Bone marrow-derived dendritic cells downregulate the progression of tumour cells (Becker,1993a,b). In the mammalian hippocampus microglial cells may stimulate T-helper cells (Frei et al., 1987;Matsumoto et al., 1986). In the case of the lizard medial cortex, microglial cells may also play a role as antigen-presenting cells (Hickey and Kimura, 1988; Streit et al., 1988). The absence of these immunoactivator cells during "reactive neurogenesis" may be a permissive factor for recruitment, maturation, and differentiation of the set of newly produced

58

LOPEZ-GARCIAET AL.

Fig. 5.


59

neurons in the ependyma. Otherwise, an immediate immunoresponse against progression of newly produced neurons may be triggered by microglia cells resident there. Temporal suppression of macrophage and microglial activity enhances axonal regeneration (Thanos et al., 19931, perhaps by an unknown action, on ganglion cell survival rather than on the axonal growth itself.

population of intraventricular macrophages (Chamberlain 1974; Ling et al., 1985b). Supraependymal cells, epiplexus cells, and free cells in the ventricles (Kolmer, 1921) are mainly phagocytic cells (Bleier, 1977a; Bleier et al., 1975; Carpenter et al., 1970; Ling et al., 1985a), with the exception of some supraependymal cells located in discrete areas of the third ventricle which have been identified as neurons (Card and Mitchell, 1978). Supraependymal phagocytic cells are thought to derive from mononuclear phagoRe-appearance of Microglia and cytes or circulating monocytes (Bleier, 1977b) and perNeo-synaptogenesis haps also from ependymal cells or from microglia-like An explanation for the re-appearance of microglial cells within the brain (Mestres and Breipohl, 1976; Sturcells in the regenerating lizard cortex may come from rock, 1978). the proposed role of microglial cells in controlling the It has been suggested that supraependymal cells are removal of synaptic contacts (“synapticstripping”)(Blinz- engaged in processing the debris that results from changinger and Kreutzberg, 1968; Kreutzberg, 1993)and neo- ing metabolic activities of ependymal cells (Bleier,1977b). Thus, it is likely that they are involved in the late cleansynaptogenesis. The axonal circuitry in the medial cortex must suffer ing of cytoplasmatic blebs with lipofuscin bodies accudramatic changes (including denervation and sprout- mulating in the apical pole of ependymal cells. Another ing phenomena) during the lesion-regeneration process. possibility is that they are also engaged in the control The late exuberant presence of microglia cells in the (downregulation) of proliferation of ependymal germiaxonal projection fields of the regenerated medial cor- native cells. tex, i.e., the zinc-rich fields which were microglia free in normal conditions (Castellano et al., 1991a), suggests a possible role for microglia in regulating synaptic plasACKNOWLEDGMENTS ticity and neo-synaptogenesis of the s o n s emitted by the newly generated granule neurons (Lopez-Garcia et We thank Prof. M. Lafarga, Prof. M. Nieto-Sanpedro, al., 1990a). and Prof. G. Raisman for their suggestions on an earlier A similar phenomenon must occur in the receptive version of the manuscript and Mr. Robin Rycroft for field of the medial cortex, i.e., its plexiform layers re- linguistic advice. This study has been financed by DGIceive the lateral cortex olfactory projection (“lizard per- CyT(PB 90 0422-C02), IVEI and GU-1037/93 grants. forant path”) plus ipsi- and contralateral axons of pyramidal neurons (Lopez-Garciaet al., 1992a).These areas are devoid of microglia in normal lizards (Castellano et REFERENCES al., 1991a), whereas they display an exuberant microglial population in regenerating lizards. Relationship Between Supraependymal Cells and Microglia The similar morphology of supraependymal cells and microglial cells and the increased presence of supraependymal cells in the late phase of regeneration suggest a possible relationship between these two types of cells (Cammermeyer, 1965; Sturrock, 1978). The embryonal intraventricular system of rats treated with the antimetabolite 6-aminonicotinamide has an increased

Fig. 5. NDPase staining after different survival times following 3-acetylpyridine injections A: Control non-injected lizard; observe microglial cells lining the cell layer of the medial cortex (arrows). B: One day atter 3AP injection; observe that there are no labelled cells in the medial cortex; nevertheless blood vessels appear labelled. C: Idem, same section as in B, showing hypothalamic preoptic area. D: Six days after 3AP injection; no cells appear labelled in the medial cortex. E: Idem, same section as in D, showing hypothalamus. F: Fifteen days after 3AP injection; labelled cells (arrows) appear in the medial cortex. G and H: Thirty days after 3AP injection; abundant microglia labelled cells appear both in the medial cortex (G) and the dorsal-dorsomedial cortex (H). Scale bars = 100 pm in A,D,F, and H; 25 pm in E and G.

Akiyama, H. and McGeer, P.L. (1989)Microglial response to 6-hydroxydopamine-induced substantia nigra lesions. Brain Res., 489:247253. Andersson, P.B., Perry, V.H., and Gordon, S. (1991) The kinetics and morphological characteristics of the macrophage-microglialresponse to kainic acid-induced neuronal degeneration. Neuroscience, 42:201214. Auer, R., Kalimo, H., Olsson, Y., and Wieloch, T. (1985) The dentate gyrus in hypoglycemia: Pathology implicating excitotoxin-mediated neuronal necrosis. Acta Neuropathol. (Berl.),67:279-288. Becker, Y. (1993a) Success and failure of dendritic cell (DC) anticancer activity may be modulated by nitric oxide synthetase (NOS) gene expression: A hypothesis. In Viuo, 7:285-288. Becker, Y. (1993b) Dendritic cell activity against primary tumors: An overview. In Viuo, 7:187-192. Beiswanger, C.M., Roscoe-Graessle, T.L., Zerbe, N., Reuhl, K.R., and Lowndes, H.E. (1993) 3-Acetvlpvridine-induceddegeneration in the dorsal root ganglia: Involvement of small d i a m e k neurons and influence of axotomy. Neuropathol. Appl. Neurobiol., 19:164-172. Berbel, P.J., Lopez-Garcia, C., Garcia-Verdugo, J.M., Regidor, J., and Marin-Giron, F. (1981)Microglia in the cerebral cortex of Lacerta. A combined Golgi-electronmicroscopic study. Morf Norm.Patol., 5:261270. Blasco-Ibanez, J.M., Martinez-Guijarro, F.J., Lopez-Garcia, C., Mellstrom, B., and Naranjo, J.R.(1992)Narine occlusion decreases basal levels of Fos protein in the cerebral cortex of the lizard Podarcis hispanica.Neuroscience, 50:647-653. Bleier, R. (1977a) Supraependymal cells of the hypothalamic third ventricle of the Tegu lizard, Tupinambis nzgropunctatus. A scanning electron microscopic study. Biol. Cell., 29:153-158. Bleier, R. (1977b)Ultrastructure of supraependymal cells and ependyma of hypothalamic third ventricle of mouse. J. Comp. Neurot., 1 7 4 359-376.

60

LOPEZ-GARCIA ET AL.

Bleier, R., Albrecht, R., and Cruce, J.A.F. (1975) Supraependymal cells of the hypothalamic third ventricle: Identification as resident macrophages of the brain. Science, 189:292-301. Blinzinger, K.H. and Kreutzberg, G. (1968) Displacement of synaptic terminals from regenerating motor neurons by microglia cells. Z. Zellforsch. Mikrosk. Anat., 85:145-157. Boya, J., Calvo, J., and Prado, A. (1979) The origin of microglial cells. J.Anat., 129:177-186. Brodsky, F.M. and Guagliardi, L.E. (1991) The cell biology of antigen processing and presentation. Annu. Rev. Immunol., 9:707-744. Cammermeyer, J . (1965) The hypependymal microglia cell. Z . Anat. Entwickl. Gesch., 124:543-561. Card, J.P. and Mitchell, J.A. (1978) Electron microscopic demonstration of a supraependymal cluster of neuronal cells and processes in the hamster third ventricle. J. Comp. Neurol., 180:43-58. Carpenter, S.J., McCarthy, L.E., and Borison, H.L. (1970) Electron microscopic study on the epiplexus (Kolmer) cells of the cat choroid plexus. Z . Zellforsch., 110:471-486. Castellano, B., Gonzalez, B., and Palacios, G. (1984) A comparative study on NDPase and ’ITPase activities on glial cells in vertebrates and invertebrates. Trab. Inst. Cajal Inu. Biol., 75:81. Castellano, B., Gonzalez, B., Dalmau, I., and Vela, J.M. (1991a) Identification and distribution of microglial cells in the cerebral cortex of the lizard: A histochemical study. J. Comp. Neurol., 311:434-444. Castellano, B., Gonzalez, B., Jensen, M.B., Pedersen, E.B., Finsen, B.R., and Zimmer, J . (1991b) A double staining technique for simultaneous demonstration of astrocytes and microglia in brain sections and astroglial cell cultures. J . Histochem. Cytochem., 39561-568. Chamberlain, J.G. (1974) Scanning electron microscopy of epiplexus cells (macrophages) in the fetal rat brain. Am. J. Anat., 139:443447. Fagan, A.M. and Gage, F.H. (1990) Cholinergic sprouting in the hippocampus: A proposed role for IL-. Exp Neurol., 110:105-120. Ferrer, I. and Sarmiento, J . (1980a) Reactive microglia in the developing brain. Acta Neuropathol. (Berl.), 50:69-76. Ferrer, I. and Sarmiento, J . (1980b)Nascent microglia in the developing brain. Acta Neuropathol. (Berl)., 50:61-67. Finsen, B.R., Jorgensen, M.B., Diemer, N.H., and Zimmer, J. (1993a) Microglial MHC antigen expression after ischemic and kainic acid lesions of the adult rat hippocampus. Glia, 7:41-49. Finsen, B.R., Tonder, N., Xavier, G.F., Sorensen, J.C., and Zimmer, J . (199313) Induction of microglial immunomolecules by anterogradely degenerating mossy fibres in the rat hippocampal formation. J. Chem. Neuroanat., 6:267-275. Font, E., Garcia-Verdugo, J.M., Alcantara, S., and Lopez-Garcia, C. (1991) Neuron regeneration reverses 3-acetylpyridine-inducedcell loss in the cerebral cortex of adult lizards. Brain Res., 551:230-235. Fox, I.H. and Kelley, W.N. (1978) The role of adenosine and 2’-deoxydenosine in mammalian cells. Annu. Rev. Biochem., 47:655486. Frei, K., Siepl, C., Groscurth, P., Bodmer, S., Schwerdel, C., and Fontana, A. (1987) Antigen presentation and tumor cytotoxicity by interferon-gamma-treated microglial cells. Eur. J.Immunol., 17:12711278. Fujimoto, E., Miki, A,, and Mizoguti, H. (1987) Histochemical studies of the differentiation of microglial cells in the cerebral hemispheres of chick embryos and chicks. Histochernistry, 87:209-216. Garcia-Verdugo, J.M., Farinas, I., Molowny, A,, and Lopez-Garcia, C. (1986) Ultrastructure of putative migrating cells in the cerebral cortex of Lacerta galloti. J. Morphol., 189:189-197. Hickey, W.F. and Kimura, H. (1988) Perivascular microglial cells of the CNS are bone marrow-derived and present antigen in vivo. Science, 239:290-292. Jensen, M.B., Gonzalez, B., Castellano, B., and Zimmer, J. (1994) Microglial and astroglial reactions to enterograde axonal degeneration: A histochemical and immunocytochemical study of the adult rat fascia dentata after entorhinal perforant path lesions. Exp. Brain Res., 98:245-260. Kolmer, W. (1921)Uber eine eigenartige Beziehung von Wanderzellen zu den Choroidealplexus des Gehirns der Wirbeltiere. Anat. Anz., 54:15-19. Kreutzberg, G.W. (1993) Dynamic changes in motoneurons during regeneration. Restor. Neurol. Neurosci., 5:59-60. Ling, E.A. (1981) The origin and nature of microglia. In: Advances in Cellular Neurobiology, Vol. 2. S. Federoff and L. Hertz, eds. Academic Press, New York, pp. 33-82, Ling, E.A., Gopalakrishnakone, P., andVoon, F.C.T. (1985a)Epiplexus cells in the embryos ofthe turtle, Trionyx sinensis. Arch Histol. Jpn., 48:355-361. Ling, E.A., Tseng, C.Y., and Wong, W.C. (1985b) An electron microscopical study of epiplexus and supraependymal cells in the prenatal rat brain following a maternal injection of 6-aminonicotinamide. J. Anat., 140:119-129.

Lopez-Garcia, C. (1993) Postnatal neurogenesis and regeneration in the lizard cerebral cortex. In: Neuronal Cell Death and Repair. A.C. Cuello, ed. Elsevier Science Publishers, Amsterdam, pp. 237-246. Lopez-Garcia, C. and Martinez-Guijarro, F.J. (1988) Neurons in the medial cortex give rise to Timm-positive boutons in the cerebral cortex of lizards. Brain Res., 463:205-217. Lopez-Garcia, C., Soriano, E., Molowny,A., Garcia-Verdugo, J.M., Berbel, P.J., and Regidor, J . (1983) The Timm-positive system of axonic terminals of the cerebral cortex of Lacerta. In: Ramon y Cajals Contributions to the Neurosciences. S. Grisolia, C. Guerri, F. Samson, S. Norton, and F. Reinoso-Suarez, eds. Elsevier Science Publishers, Amsterdam, pp. 137-148. Lopez-Garcia, C., Molowny, A., Rodriguez-Serna, R., Garcia-Verdugo, J.M., and Martinez-Guijarro, F.J. (1988) Postnatal development of neurons in the telencephalic cortex of lizards. In: The Forebrain of Reptiles: Current Concepts of Structure and Function. W.K. Schwerdtfeger, and W. Smeets, eds. Karger, Basel, pp. 122-130. Lopez-Garcia, C., Molowny, A,, Garcia-Verdugo, J.M., Martinez-Guijarro, F.J., and Bernabeu, A. (1990a) Late generated neurons in the medial cortex of adult lizards send axons that reach the Timmreactive zones. Deu. Brain Res., 57:249-254. Lopez-Garcia, C., Molowny, A., Garcia-Verdugo, J.M., Martinez-Guijarro, F.J., Font, E., and Alcantara, S. (1990b)Neurogenesis postnatal tardia y regeneracion en la corteza cerebral de la lagartija comun. Biol. Desenvolup., 8:183-192. Lopez-Garcia,C., Molowny, A,, Martinez-Guijarro, F.J., Blasco-Ib~ez, J.M., Luis de la Iglesia, J.A., Bernabeu, A,, and Garcia-Verdugo, J.M. (1992a) Lesion and regeneration in the medial cerebral cortex of lizards. Histol. Histopathol., 7:725-746. Lopez-Garcia, C., Molowny, A,, Martinez-Guijarro. F.J., Garcia-Verdugo, J.M., and Blasco-banez, J.M. (1992b) Neurogenesis during adult cerebral cortex regeneration in lizards. Abstracts 5th Int. Congress Cell Bwl., 333 (abstract). Lowe, J., MacLennan, K.A., Powe, D.G., Pound, J.D., and Palmer, J.B. (1989) Microglial cells in human brain have phenotypic characteristics related to possible function as dendritic antigen presenting cells. J.Pathol., 159:143-149. Martinez-Guijarro, F.J., Desfilis, E., and Lopez-Garcia, C. (1990) Organization of the dorsomedial cortex in the lizard Podarcis hispanica. In: The Forebrain in Non-mammals. New Aspects of Structure and Development. W.K. Schwerdtfeger andP. Germroth, eds. SpringerVerlag, Berlin, pp. 77-92. Martinez-Guijarro, F.J., Soriano, E., Del Rio, J.A., and Lopez-Garcia, C. (1991) Zinc-positive boutons in the cerebral cortex of lizards show glutamate immunoreactivity. J . Neurocytol., 20:834-843. Matsumoto, Y., Hara, N., Tanaka, R., and Fujiwara, M. (1986) Immunohistochemical analysis of the rat central nervous system during experimental allergic encephalomyelitis, with special reference to Ia-positive cells with dendritic morphology. J.Immunol., 136:36683676. Mestres, P. and Breipohl, W. (1976) Morphology and distribution of supraependymal cells in the third ventricle of the albino rat. Cell Tissue Res., 168:303-314. Miyake, T. and Kitamura, T. (1991) Proliferation of microglia in the injured cerebral cortex of mice as studied by thiamine pyrophosphatase histochemistry combined with 3H-thymidine autoradiography. Acta Histochem. Cytochem., 24:457463. Molowny, A. and Lopez-Garcia,C. (1978)Estudio citoarquitectonicode la corteza cerebral de reptiles. 111. Localizacion histoquimica de metales pesados y definicion de subregiones Timm-positivas en la corteza de Lacerta, Chalcides, Tarentola y Malpolon. Trab. Inst. Cajal Inu. Biol., 7055-74. Moschella, S.L. and Cropley, T.G. (1990) Mononuclear phagocytic and dendritic cell systems. J.Am. Acad. Dermatol., 22:1091-1097. O’Hearn, E., Long, D.B., and Molliver, M.E. (1993) Ibogaine induces glial activation in parasagittal zones of the cerebellum. Neuroreport, 4:299-302. Ogawa, M., Araki, M., Naito, M., Takeya, M., Takahashi, K., and Yoshida, M. (1993) Early changes of macrophage-like immunoreactivity in the rat inferior olive after intraperitoneal 3-acetylpyridine injection. Brain Res., 610:135-140. Rathbone, M.P., Middlemiss, P.J., Kim, J.K., Gysbers, J.W., Deforge, S.P., Smith, R.W., and Hughes, D.W. (1992) Adenosine and its nucleotides stimulate proliferation of chick astrocytes and human astrocytoma cells. Neurosci. Res., 13:l-17. Rio Hortega, P. (1932) Microglia. In: Cytology and Cellular Pathology of the Nervous System, Vol. 2. W. Penfield, ed., P.B. Hoeber, New York, pp. 481-534. Sagar, S.M., Sharp, F.R.. and Curran. T. (1988) Exmession of c-fos protein in brain: Metabolic mapping at the cellular level. Science, 240:1328-1331, Sanyal, S. and De-Ruiter, A. (1985) Inosine diphosphatase a s a his-

MICROGLIA AND LIZARD CORTEX REGENERATION tochemical marker of retinal microvasculature, with special reference to transformation of microglia. Cell Tissue Res., 241:291-297. Shaw, J.A.G., Perry, V.H., and Mellanby, J . (1990) Tetanus toxininduced seizures cause microglial activation in rat hippocampus. Neurosci. Lett., 120:66-69. Sprecher, E. and Becker, Y. (1993) Role of Langerhans cells and other dendritic cells in disease states. In Viuo, 7:217-228. Stensaas, L.J. (1983) Regeneration in the spinal cord of the newt Nottophtalmus (Triturus) pyrrogaster. In: Spinal Cord Reconstruction. C.C Kao, R.P. Bunge, and P.J. Reier, eds. Raven Press, New York, pp. 121-149. Stenwing, A.E. (1972) The origin of brain macrophages in traumatic lesions, Wallerian degeneration and retrograde regeneration. Neuropathol. Exp. Neurol., 31:696704. Streit, W., Graeber, M., and Kreutzberg, G. (1988) Functional plasticity of microglia: A review. Glia., 1:301-307. Sturrock, R.R. (1978) A developmental study of epiplexus and supraependymal cells and their possible relationship to microglia. Neuropathol. Appl. Neurobiol., 4:307-322.

61

l l a n o s , S.,Mey, J., and Wild, M. (1993) Treatment of the adult retina with microglia-suppressing factors retards axotomy-induced neuronal degradation and enhances axonal regeneration in uzuo and in vitro. J.Neurosci., 13:455-466. Turner, J.E. and Singer, M. (1975) The ultrastructure of Wallerian degeneration in the severed optic nerve of the newt. Anat. Rec., 181:267-286. Turner, J.E. and Glaze, K.A. (1977) The early stages of Wallerian degeneration in the severed optic nerve of the newt (Triturus uiriolescens).Anat. Rec., 187:291-310. Wilson, M.A., Gaze, R.M., Goodbrand, I.A., and Taylor, J.S.H. (1992) Regeneration in the Xenopus tadpole optic nerve is preceded by a massive macrophagdmicroglial response. Anat. Embryol., 186: 15-89. Zammit, P.S., Clarke, J.D.W., Golding,J.P., Goodbrand, I.A.,and Tonge, D.A. (1993) Macrophage response during axonal regeneration in the axolotl central and peripheral nervous system. Neuroscience, 54: 781-789.