Immunological cross-reactivity of cultured rat hippocampal neurons with goldfish brain proteins synthesized during memory consolidation

Brain Research, 386 (1986) 245-257 Elsevier

245

BRE 12117

Immunological Cross-Reactivity of Cultured Rat Hippocampal Neurons with Goldfish Brain Proteins Synthesized during Memory Consolidation RUPERT SCHMIDT1, FRIDOLIN LOFFLER2, HANS WERNER MI]LLER3 and WILFRIED SEIFERT4

l lnstitut far Zoologie, J. W. Goethe-Universitiit Frankfurt, Frankfurt, 2physiologisch-chemisches lnstitut der Universitiit Wiirzburg, Warzburg, 3Neurologische Klinik, Universitiit Dasseldorf, Dasseldorf and 4Abteilung fiir Neurobiologie, Max-Planck-lnstitut ffir Biophysikalische Chemie, G6ttingen (F. R. G. ) (Accepted 1 April 1986)

Key words: Rat hippocampus - - Memory and learning - - Goldfish brain protein - - Behavioural plasticity - Immunohistofluorescence staining - - Embryonic glycoprotein - - Plasticity of the CNS - - Pyramidal cell culture

Ependymins are goldfish brain glycoproteins exhibiting a specifically enhanced rate of synthesis when the animals adopt a new pattern of swimming behavior. With specific antisera against ependymins it has become possible to look for ependymin-like immunoreactivity in other animal species, both qualitatively by immunofluorescence staining and quantitatively by radioimmunoassay. Ependyrain-like immunoreactivity was detected not only in other fish but also in rat brain. In the rat radioimmunoassay measurements were highest for the hippocampal formation and for cultured neurons derived from the embryonic hippocampus. Immunofluorescence staining was performed on various cell culture systems derived from rat brain, in order to establish which cell type contains the antigen. Only neuronal cell populations reacted with the anti-ependymin antisera. Cells derived from embryonic rat brain hippocampus which resembled pyramidal neurons stained particularly bright for ependymin-like immunoreactivity. The antigenic material was distributed throughout the cytoplasm including the neuronal extensions. Various neuron-specific antisera have been used to counterstain the cells containing ependymin-like immunoreactivity. INTRODUCTION Results from many laboratories indicate that the consolidation of long-term m e m o r y involves the biosynthesis of brain proteins (for review see refs. 2, 12, 33, 48, 49 and 51). Using radioactive double-labeling techniques 3 specific goldfish brain proteins, a, fl and ~, (molecular weights 37, 32 and 26 kDa), have been identified, which exhibited an enhanced rate of synthesis when the animals adopted a new pattern of swimming behavior 45. The metabolic change was confined to the brain cytoplasm and extracellular fluid including cerebrospinal fluid 47 and was only observed in learning animals. The fl and 7 proteins are acidic glycoproteins, which were purified and used to immunize rabbits 46. U p to 24 h after training the specific antibodies impaired long-term m e m o r y formation when they were injected into goldfish brain ventricles, but they were without influence on short-term

memory or the performance of the swimming behavior itself 37'38,4°'5°. By immunohistofluorescence techniques the fl and 7 proteins were localized to cells of the periventricular gray of the ependymal zone and neurons in the optic tectum and the vagal lobes of untrained goldfish 4'35'38. According to their distribution they were named 'ependymins'. Cells from the ependymal zone of the goldfish optic tectum were grown in culture and have been demonstrated to synthesize ependymins de novo and to secrete them into their extracellular environment e3. Physiologically ependymins occur mainly as dimers 41, but dissociate into their monomeric forms in the extracellular fluid. A n extracellularly located protease activity proteolytically modifies ependymin fl to become the y polypeptide chain 42. The development of a sensitive and specific radioimmunoassay for the quantitative analysis of goldfish ependymins 41 has enabled us to look for cross-reac-

Correspondence: R. Schmidt, Department of Zoology, J.W. Goethe-University, Siesmayerstrasse 70, Postfach 111932, D-6000 Frankfurt am Main, F.R.G, 0006-8993/86/$03.50 (~) 1986 Elsevier Science Publishers B.V. (Biomedical Division)

246 tive substances in higher vertebrates. We demonstrate here, that ependymin-like immunoreactivity is not only expressed in other fish but also in rat brain. The highest measurements of antigenic material were obtained for cultured neuronal cells derived from embryonic rat hippocampus. Morphologically these cells resemble pyramidal neurons and they also stain very brightly with anti-ependymin antisera used in indirect immunohistofluorescence experiments. The occurrence of ependymin-like immunoreactivity in the hippocampal formation is remarkable because experimental data obtained with mammals and clinical observations on humans point towards a pivotal role of the hippocampus in memory formation 24'26'29'43'53. Preliminary accounts of some of the results have previously been published in abstract form34.36.39. MATERIALS AND METHODS

Animals Goldfish, Carassius auratus (Common and Comet varieties), and other fish species were obtained from local pet shops, A. J~iger, Aquaristik-Bedarf, Marburg (F.R.G.). Torpedo marmorata was supplied from the Institut de Biologie Marine, Arcachon (France). Toads, Bufo bufo, were delivered by R. Stein, Lauingen (F.R.G.), Xenopus was grown in the Institute of Zoology at Frankfurt (F.R.G.). NMRI mice and Wistar rats were obtained from Dr. Henschler, Institute of Toxicology, University of Wfirzburg (F.R.G.) and from Ivanovas, Kissleg

(F.R.G.). Chemicals Fetal calf serum and horse serum were obtained from Boehringer, Mannheim (F.R.G.). N,N-dimethylformamide, Folin and Ciocalteu's phenol reagent and fl-mercaptoethanol were from Merck, Darmstadt (F.R.G.). lZSI-monoiodinated 3-(4-hydroxyphenyl)propionic acid N-hydroxysuccinimide ester (Bolton and Hunter reagent), specific activity 2000 Ci/mmol, was from Amersham Buchler, Braunschweig (F.R.G.). Concanavalin A, covalently bound to Sepharose 4B, PD-10 columns, prepacked with Sephadex G-25M, and Sephadex G-75 were purchased from Pharmacia Fine Chemicals AB, Uppsala (Sweden). Dulbecco's modified Eagle's medium

(DMEM, Gibco) was obtained from C. Roth, Karlsruhe (F.R.G.). K-penicillin G and streptomycin sulfate were bought from Serva, Heidelberg (F.R.G. t Bovine serum albumin, crystallized, Coomassie brilliant blue R, gelatin, hydrocortisone, insulin (from bovine pancreas), poly-L-lysine, a-methyI-D-mannoside, grade 1II, progesterone, putrescine dihydrochloride, sodium dodecyl sulfate (SDS), human transferrin and triiodothyronine were from Sigma Chemical Co., Munich (F.R.G.). Poly-L-ornithine-HBr, code No. 71-129, was obtained from Miles Laboratories, Frankfurt (F.R.G.).

Cell culture methods Several different methods were applied to grow glial and neuronal cells from various sources in culture: glial cells from brains of neonatal Wistar rats were obtained by mechanical dissociation 55, A detailed method for obtaining primary neuronal cell cultures has been described elsewhere 2°. Briefly, whole brains were removed from rat embryos at 15 days of gestation and dissociated mechanically. Some 3 x 106 viable cells (viability >75%) were seeded in 5 ml of Dulbecco's modified Eagle's medium (DMEM), supplemented by 10% horse serum, Kpenicillin (20 U/ml) and streptomycin sulfate (20 ~tg/ml) in poly-L-ornithine-coated Petri dishes 1. After 24 h, when all cells were attached to the plates, the medium was removed, the cells were washed once with 5 mi DMEM, and subsequently 3.3 ml of DMEM were added which had previously been conditioned by rat glial cell cultures for two days and had then been supplemented by the ingredients of Bottenstein's N2-medium ~. Primary neurons were obtained from embryonic rat hippocampus at 18 days of gestation, when the pyramidal neurons (but not yet the granule cells) have become postmitotic. They were cultured using the method of Peacock et al. 31 with the following modification. The dissociated cells were grown for 4 days on poly-lysin-coated coverslips (30 mm in diameter) in serum-free DMEM. The culture medium was conditioned by primary glial cell cultures from rat cerebrum and supplemented with hormones according to Bottenstein and Sato 7. This primary culture is highly enriched in neurons of pyramidal morphology containing less than 10% of other cell types 4a. The primary cultures enriched in glial cells which

247 were used to condition the DMEM for the hippocampal neurons were also prepared from embryonic rat brain at 18 days of gestation. Cells were dissociated with trypsin-EDTA and single cells were dispersed in DMEM supplemented with 20% heat-inactivated fetal calf serum and plated in 10 cm plastic dishes for culturing. The cultures were incubated at 37 °C in humidified atmosphere (95% air, 5% CO2) for 8 days27.

Antisera Normal rabbit serum (young, type 1, non-sterile select, non-hemolyzed) was from Pel-Freez Biologicals, Inc., Rogers (AR, U.S.A.). Lyophilized goat serum directed against rabbit immunoglobulins G (IgG, heavy and light chains) was purchased from Cappel Laboratories, Inc., Cochranville (PA, U.S.A.). Antisera against glial fibrillary acidic protein (GFAP), an intracellular marker for astrocytes, produced in guinea pigs were obtained from K. Hailermayer, Wfirzburg (F.R.G.), and from A. Bignami, Boston (U.S.A.) 5'14. Anti-neuron-specific-enolase (NSE, 2-phospho-D-glycerate hydrolase, EC 4.2.1.11) antiserum produced in rabbits was a generous gift from P.G. Marangos, Bethesda (U.S.A.), and the monoclonal antibody against A4, a neuronal cell surface protein 1°, was a kind gift by J. Cohen, London (England). The monoclonal antibody A2B5 directed against the neuronal ganglioside GQ 13 was obtained from Seralab, London. Oligodendrocytes were identified by a monoclonal antibody directed against galactocerebroside (mGalC, ascites fuid), a specific cell surface marker for oligodendrocytes and Schwann cells32. Goldfish brain fl and y proteins were isolated and used to produce antisera in rabbits as described in detail41. In brief, the two glycoproteins were isolated from goldfish brain cytoplasm (or alternatively from goldfish brain extracellular fluid) by lectin chromatography on Sepharose-bound concanavalin A. The mixture of the two proteins which contains mainly the dimeric conformations of the proteins was either used directly for immunization or was further separated by SDS polyacrylamide gel electrophoresis (PAGE) to give the monomeric fl and ~' proteins which were eluted electrophoretically from the gels. Both, preimmune sera from the same rabbit and antisera from which the specific antibodies against fl and

7 had been removed were used as controls. The specific antibodies were removed from the antisera by incubation with the appropriate antigen (1 h at 37 °C and 48 h at 4 °C) followed by sedimentation of the antibody-antigen complex (see ref. 42 for details). The following second antibodies were used: Fluorescein-conjugated goat IgG against rabbit IgG (FITC anti-rabbit IgG), fluorescein-conjugated goat IgG against guinea pig IgG (FITC anti-guinea pig IgG), rhodamine-conjugated goat IgG against mouse IgG (TRITC anti-mouse IgG) and rhodamineconjugated goat IgG against rabbit IgG (TRITC anti-rabbit IgG), all from Mallinckrodt (Nordic), Dietzenbach (F.R.G.). For double-staining experiments, the fluorochrome conjugates were preabsorbed with either mouse, rabbit or guinea pig IgGs to remove cross-reactivity with the inappropriate antibody.

Immunofluorescent staining Various methods for indirect immunofluorescent staining were tried and compared. For intracellular antigens in cultured cells best results were obtained in the following way. The coverslips with the attached cells were washed 5 times in DMEM containing 1% bovine serum albumin (DMEM-BSA), mildly fixed in 5% acetic acid in ethanol (15 min at -20 °C) and rehydrated by dipping them into water for 30 s. This procedure also opens the intracellular compartments and makes them accessible to antibodies. The cells were then incubated with the first antibody (diluted 1:100 in DMEM-BSA) for 30 min at room temperature, washed 5 times in DMEM-BSA and incubated with the second antibody (fluorescence-labeled, diluted 1:20 in DMEM-BSA) for another 30 min at room temperature. The coverslips were then washed again 5 times in phosphate-buffered saline (PBS) and mounted upside down on microscopic slides in a 1:1 mixture of glycerol and PBS (pH 8.0). In some experiments the staining protocol of L6ffier et ai. 2° was used: cells were opened by fixation in 3.5% formaldehyde in PBS at room temperature, washing steps were done in PBS and incubations with the antisera in PBS containing 0.3% Triton X-100 and 50% normal goat serum (60 min, dilutions as indicated above). Results obtained with the 'formaldehyde-Triton-procedure' were similar to those obtained after fixation in acetic acid-ethanol.

248 F o r surface m a r k e r proteins basically the same p r o c e d u r e was used, but the fixation (and rehydration) before the first a n t i g e n - a n t i b o d y reaction was omitted. Instead, the cells were fixed for 5 min in 3.5% f o r m a l d e h y d e in PBS after washing off the excess of the first a n t i b o d y or in 5% acetic acid in ethanol at - 2 0 °C for 15 min after washing off the excess of the second antibody. They were then washed thoroughly and m o u n t e d . The p r e p a r a t i o n s were either viewed in a Leitz Orthoplan, in an O l y m p u s IMT-2 or in a Zeiss fluoresence microscope e q u i p p e d with the a p p r o p r i a t e filter combinations. P h o t o m i c r o g r a p h s were taken on K o d a k Tri-X-pan 135 or E k t a c h r o m e 160.

Radioimmunoassay F o r quantitative analysis culture m e d i a from cell culture systems were collected and the cell m o n o l a y ers were rinsed twice with ice-cold PBS and d e t a c h e d from the culture dishes ( c e r e b r u m ) or from the cov-

erslips (hippocampus) with a r u b b e r policeman into 0.16 M NaCI. Cells were pelleted by centrifugation at 200 g for 10 min, resuspended in 10 mM tris(hyd r o x y m e t h y l ) - a m i n o m e t h a n e - H C 1 buffer, containing 0.2 m M MgC12, left on ice for 15 rain to induce swelling, and then r u p t u r e d in a D o u n c e homogenizer. Cell h o m o g e n a t e s were immediately frozen and stored at - 7 0 °C prior to the r a d i o i m m u n o a s s a y and protein determination. Immunological cross-reactivity with goldfish brain ependymins was m e a s u r e d by a very sensitive radioimmunoassay capable of detecting f e m t o m o l a r quantities of these proteins 41. This assay is highly selective for e p e n d y m i n s and responds only to the m o n o m e r i c form of the proteins. It does not differentiate, however, between e p e n d y m i n / 3 and ~,41,42. Prior to the m e a s u r e m e n t s all samples were d e n a t u r e d by heating (3 min at 100 °C) in o r d e r to expose the antigenic determinants. O n e h u n d r e d microliters of each sample were incubated with 125I-labeled e p e n d y m i n / 3 or

TABLE I

Cross-reactivity with goldfish brain ependymins Cross-reactivity is expressed as percentage of the total protein content in the fraction contributed by ependymin-like immunoreactivity from (n) experiments + S.E.M. ECF, Extracellular brain fluid including cerebrospinal fluid.

Species

Fluid~cells

% + S. E.M.

Catfish ( Ictalurus nebulosus) Goldfish (Carassius auratus)

ECF ECF cytoplasm ECF cytoplasm ECF cytoplasm cytoplasm ECF cytoplasm cytoplasm ECF cytoplasm larval cytoplasm ECF cytoplasm ECF total brain hippocampus frontal lobe cerebellum tegmentum medulla oblongata primary brain cell cultures glial cell cultures ependymal cell cultures hippocampal neuronalcellcultures

1.04 13.62 + 1.30 5.60 + 0.46 0.003 0.07 12.69 6.62 0.009 0.016 0.079 + 0.007 0.45 0.06 0.029 + 0.008 0.074 0.04 + 0.01 0.009 +_0.004 0.03 _+0.02 0.016 + 0.008 ().034 +__0.011 0.006 0.003 0.005 0.011 0.007 0.025 + 0.015 0.011 _+0.003 1.3 + 0.9

Gouramy (Helostoma temnicky ) Guppy ( Poecilia reticulata) Rudd ( Scardinius erythrophthalrnus) Topminnow (Aplocheilus lineatus) Torpedo (Torpedo marmorata) Xenopus (Xenopus laevis) Toad ( Bufo bufo) Mouse ( Mus) Rat ( Rattus) cytoplasm derived from cytoplasm derived from cytoplasm derived from cytoplasm derived from cytoplasm derived from cytoplasm derived from cells derived from cells derived from cells derived from cells derived from

(n) (1) (106) (68) ( 1) ( 1) (1) {l ) ( 1) (t) (6) ( 1) ( 1) (3) (1) (5) (4) (2) (4)

(3) ( 1) (1) ( 1) (1) ( 1) (4) {2) (3)

249 7 and rabbit anti-ependymin antiserum at a titer of 1:4700 as described previously41. The bound antigen was precipitated by the double antibody technique, using goat anti-rabbit IgG as second antiserum. Each sample was measured at three different dilutions, each in duplicate.

Protein determination The protein content of the samples was measured by a modification 19 of the method of Lowry et al. 21 using bovine serum albumin as a standard protein.

Polyacrylamide gel electrophoresis SDS-PAGE on 10% polyacrylamide gels TM was used to determine, whether proteins comigrating with goldfish brain ependymins can be isolated from rat brain. For this purpose, glycoproteins containing an a-glycosidic bonding were isolated by affinity chromatography on Sepharose-bound concanavalin A from rat brain extracellular fluid or cytoplasm as described for goldfish ependymins (see above). Samples were reduced by boiling with fl-mercaptoethanol and a small amount of 125I-ependymin fl or ~ was added in order to identify the authentic migratingpositions for ependymins. Part of the gels was cut into 1 mm strips and counted in a Philips gamma counter (model PW 4800), part was fixed in 12.5% trichloroacetic acid and stained with Coomassie blue in 9% acetic acid in 50% aqueous methanol. Pilot experiments had shown, that the amount of added radioactive ependymin (1 pmol) was too small to stain with Coomassie blue. RESULTS

Occurrence of ependymin-like immunoreactivity in various animal species Ependymins have been first identified in goldfish being highly enriched in brain cytoplasm and brain extracellular fluid. With the radioimmunoassay comparably high concentrations have been determined in another toothless carp, the rudd Scardinius erythrophthalmus (Cyprinidae). The amount of immunological cross-reactivity measured in the brains of fish from other families including catfish, guppy, gouramy, a minnow and the electric ray Torpedo, however, was considerably smaller (Table I). Ependyminlike immunoreactivity determined in species from

other classes like amphibians (Pipidae and Bufonidae) and mammals was even lower (Table I).

Radioimmunoassay measurements on rat brain samples The amount of immunological cross-reactivity with goldfish ependymins has been determined in the total rat brain and in various brain regions (Table I). Generally, the observed cross-reactivity was rather low, but measurements obtained for the hippocampal formation were higher than those of other brain regions or the total brain homogenate. Immunological crossreactivity has also been analysed in isolated cells obtained from cell cultures (Table I). Again the measured immunological cross-reactivity was most pronounced in neuronal cells obtained from embryonic hippocampus.

Polyacrylamide gel electrophoresis Fig. 1 shows rat glycoproteins isolated from brain cytoplasm and separated by SDS-PAGE. The faint protein band marked P26 is comigrating with goldfish brain ependymin 7, the band marked P52 is comigrating with the dimeric ependymin Y2. No protein band could be identified at the position of goldfish ependymin fl (marked 32). The protein bands comigrating with ependymins were cut out from the gels and

F

OAD

RAT

52........................................ ii:ill

d )mlZSIx10 -4

I

32~ ~" 2 6 ......

ili!i~il

o'

~

Fig. 1. SDS-PAGE of toad and rat brain glycoproteins. Cytoplasmic brain glycoproteins were isolated by concanavalin A affinitychromatography, separated by SDS-PAGE and stained with Coomassie blue. Approximately 1 pmol of 12SI-labeled goldfish ependyminy was added to the protein mixture in order to identifythe authentic migrating position of the y-polypeptide by counting the y-ray emission (right side). Arrows indicate the positions of P26 and P52. No protein could be identified at the migrating position of goldfish ependyminfl (32). Phosphorylase B (94 kDa), bovine serum albumin (68 kDa), ovalbumin (45 kDa), carbonic anhydrase (30 kDa), soybean trypsin inhibitor (21 kDa), and lysozyme (14.3 kDa) were used as molecular weight markers. Arrowheads mark the position of the migrating dye fronts.

250 eluted by electrophoresis (see Shashoua 45 for methodological details). A f t e r further purification by dialysis the isolated proteins were tested for immunological cross-reactivity in the r a d i o i m m u n o a s s a y . The

protein comigrating with e p e n d y m i n ~ showed 5,5%: cross-reactivity with goldfish brain e p e n d y m i n 7 (i.e. 1000 /~g of rat P26 displaced as much radioactive goldfish e p e n d y m i n from rabbit anti-goldfish epen-

Fig. 2. Cytochemical localization of ependymin-like immunoreactivity. Primary neuronal cell cultures were prepared from embryonic rat brains at 15 days of gestation and kept in culture for 10 days. Intracellular double staining for ependymins (a) and GFAP (e). c: phase contrast view corresponding to a and e. Extracellular double staining for ependymins (b) and GFAP (f). d: phase contrast view corresponding to b and f. Cells were fixed in formaldehyde-Triton. Anti-ependymin 3, antibodies were visualized by TRITC anti-rabbit IgG and anti-GFAP antibodies by FITC anti-guinea pig IgG. Bars represent 15/~m.

251 dymin antibodies as 55/~g of the authentic goldfish ependymin). None of the other rat brain proteins, which were isolated for control purposes, demonstrated any cross-reactivity with the ependymins. For glycoproteins isolated from toad brain, a very similar result was obtained. Only one protein comigrating with goldfish ependymin 7 exhibited 11% cross-reactivity in the radioimmunoassay.

Immunohistofluorescence staining of rat brain cryostat sections Coronal sections (10/tin) of adult and embryonic rat brains were used for indirect immunofluorescence staining with rabbit anti-ependymin antisera (titer 1:100) and FITC-labeled goat anti-rabbit IgG antisera. Unfixed sections and sections fixed in ethanolic acetic acid or formaldehyde were compared (see Materials and Methods). Although some staining of cells was observed in the hippocampal formation, the contrast was poor in all instances (sections not shown). We have tried, therefore, to obtain clearer results with cell cultures, where the individual cells are better exposed to the antisera and a distinction between different cell populations can be made more easily. Immunofluorescence double staining of primary rat brain cultures Primary neuronal cell cultures from embryonic Wistar rat brains (15 days of gestation) were fixed with formaldehyde-Triton and stained with rabbit anti-ependymin antisera followed by TRITC antirabbit IgG (Fig. 2a). Only neuronal cells stained for ependymin-like immunoreactivity. Astrocytes which are the major non-neuronal contamination in this culture were counterstained with guinea pig antiGFAP, followed by FITC anti-guinea pig IgG (Fig. 2e). The two antisera clearly stained different cell populations. The neuronal nature of the cells demonstrating ependymin-like immunoreactivity was further confirmed by staining them with rabbit anti-NSE and guinea pig anti-GFAP antiserum. In these independent experiments (cf. ref. 20) the same cell population stained for NSE which also stains for ependymin-like immunoreactivity. Immunohistofluorescence double staining with anti-ependymin and antiNSE antibodies was not feasible, because both antisera were from rabbit.

In a parallel set of experiments, it was tested whether cells would stain for ependymin-like material on their surface. For this purpose, cells were first incubated with anti-ependymin antiserum and then fixed with formaldehyde (Fig. 2b, d and f). Only faint staining of some neuronal cells was observed (Fig. 2b). It is possible, that plasma membranes of some neurones in these cultures had become partially permeable during incubation, or that the cells were secreting ependymins. As anticipated, anti-GFAP antisera did not reveal staining of cell membranes (Fig. 2f). Various rabbit antisera directed against goldfish brain ependymins were compared. The strongest immunoreactivity was observed with an antiserum directed against the immunogen ependymin 7 isolated from goldfish cytoplasm. Less prominent reactions were obtained with antisera directed against ependymins isolated from the extracellular fluid, against the dimeric conformations of ependymins or against ependymin fl, which is the physiological precursor molecule of ependymin 7 in goldfish42. This is in concordance with the notion, that a cross-reactive glycoprotein comigrating with ependymin y was observed on PAGE, but no protein comigrating with ependyrain fl (see above). It should be noted, however, that all antisera directed against the different ependymins and their different conformations exhibit a large degree of cross-reactivity. Primary neuronal cell cultures and glial cell cultures derived from neonatal Wistar rat brains or from newborn mice did not stain with anti-ependymin antisera (now shown).

Immunofluorescence staining of hippocampal neurons By radioimmunoassay the highest amount of immunological cross-reactivity with goldfish ependymins in the rat brain was measured in hippocampal neurons. Therefore cell culture techniques were used in an attempt to identify which specific cell population contains ependymin-like immunoreactivity. Dispersed cells from embryonic rat brain hippocampus (18 days of gestation) were grown as a monolayer on poly-lysine-coated coverslips. This culture contains almost exclusively neuronal cells, i.e. more than 90%, and they are supported with a neurotrophic factor by a primary glial cell culture seeded un-

I'J f,J'l

253 derneath the coverslips 27'2s. Four different staining protocols were compared, using either formaldehyde-Triton or ethanolic acetic acid for fixation (see Materials and Methods for details). The obtained staining was similar, but fixation by ethanolic acetic acid resulted in better cell preservation and higher contrast. Fixation was either done before exposure to the first antibody (intracellular staining) or after antibody treatment (surface markers). All neurons which exhibited the morphological characteristics of pyramidal cells (90% of all cells in the culture) stained brightly for ependymin y (Fig. 3). The antigenic material was located throughout the cytoplasm including all the dendritic processes. Cell nuclei remained unstained (Fig. 3e). It will be noted that the staining was most prominent in areas where connections had formed between the processes, which resembled synapses (arows in Fig. 3). Several different antisera directed against neuronspecific marker substances were used to counterstain the cells, including a monoclonal antibody against the neuronal cell surface protein A4 (ref. 10) (see Fig. 4) and the monoclonal antibody A2B5 directed against the neuron-specific ganglioside GQ (ref. 13) (not shown). These two antibodies counterstained all cells with ependymin-like immunoreactivity, but neither astrocytes nor oligodendrocytes. Contaminating oligodendrocytes were identified by a monoclonal antibody against GalC, galactocerebroside, a surface characteristic of oligodendrocytes and Schwann cells32. There was no overlap in ependymin-like and GalC immunoreactivity (not shown). In another series of experiments 14 different antiependymin antisera were compared for their staining characteristics against embryonic hippocampal neurons. As for primary neuronal cultures (see above), best results were obtained with the antiserum raised against cytoplasmic ependymin y as immunogen.

Strong immunofluorescence was also observed with antisera against the dimeric ependymins (/3), and ),2) and some with anti-cytoplasmic ependymin ft. The following control sera were used: preimmune sera from the same rabbits and immune sera from which the specific anti-ependymin antibodies had been removed by incubation with an excess of the appropriate antigen and sedimentation of the antibody-antigen complex. No staining was observed in these instances. One antiserum had been prepared against a mixture of the dimeric ependymins which were presumably contaminated with their monomeric subunits. Like the other anti-ependymin antisera, this serum reacted only with neuronal cells of pyramidal morphology, the staining pattern, however, was somewhat different. Whilst the cytoplasmic staining after fixation in ethanolic acetic acid resembled that obtained with the other antisera, it was the only serum giving also strong labeling of the cell surfaces, when cells were treated with the first and second antiserum and fixed after removal of excessive antibodies (Fig. 3f and 4). The immunofluorescence labeling of the plasma membrane distributed on the cell body as well as on the processes. The surface labeling cannot be explaind by artificial penetration of the antibodies through permeable cell membranes, since the viability of the cells was monitored with the trypan blue exclusion test (cp. also ref. 28). The labeled cells have been identified as neurons by means of a cell type specific antibody against the protein A4. All neurons stained for ependymin-like immunoreactivity. Non-labeled non-neuronal cell types, when contaminating the hippocampus cell culture (as indicated by the arrow in Fig. 4a), have been used as internal controls for the staining specificity of the anti-ependymin antibody. Control experiments with rabbit preimmune serum and mouse ascites fluid showed no immunofluorescence (Fig. 4d and f). Also these obser-

Fig. 3. Immunohistofluorescence staining of hippocampus pyramidal neurons with anti-ependymin antisera. Neurons were derived from embryonic rat brains (16th day of gestation) and kept in culture for 3 days. a-e: for intracellular staining, cells were fixed and opened with ethanolic acetic acid and incubated with anti-ependymin y. FITC-conjugated goat anti-rabbit IgG was used as fluorochrome. All neurons with pyramidal appearance (see inset in a) stained brightly for ependymin-like immunoreactivity. Astrocytes (A in a) exhibited only weak background staining. The immunoreactivity in neurons was distributed throughout the cytoplasm, including all processes (e), but cell nuclei remained unstained (d). Small arrows point to connections between neurons which resembled synapses (c). f: for membrane staining, cells were incubated with anti-ependymin antiserum against the dimeric conformation of the protein first and fixed after removal of the second antibody (FITC-conjugated goat anti-rabbit IgG). Staining is seen in the form of patches covering the whole pyramidal neuron including cell processes. Bars represent 5/tm.

254

Fig. 4. Double immunofluorescent labeling of the cell surface in cultures from embryonic rat hippocampus. Cells on coverslips ( lSth day of gestation, 3 days in culture) were incubated sequentially for 30 min at room temperature in a humidified chamber with antiependymin antiserum directed against the dimeric conformation, fluorescein-conjugated anti-rabbit IgG, anti-A4 and finally rhodamin-conjugated goat anti-mouse IgG. Between the incubations with the different antibodies the cells were washed by immersion through 5 changes of DMEM containing 1% BSA. After the last wash the cells were fixed with ethanolic acetic acid. c: fluorescence labeling with anti-ependymin, e: fluorescence labeling with monoclonal antibody against A4. a: phase contrast photomicrograph of the hippocampal cell culture stained in c and e. The arrow in a points to a non-neuronal cell, probably an astrocyte, not labeled by the antibodies, d: control staining using preimmune rabbit serum for the first incubation, f: control staining using mouse ascites fluid for the first incubation, b: phase contrast photomicrograph corresponding to d and f. Bars represent l%~m

255 vations exclude any penetration of the neurons with immunoglobulins prior to the permeabilization by fixation. DISCUSSION The development of highly specific antisera against ependymins has enabled us to look for ependymin-like immunoreactivity in various animal species. Besides of cyprinids and other fish families, ependymin-like immunoreactivity was also observed in xenopus, toad and especially in the rat brain hippocampus. The hippocampal formation is part of the archipallium, first developed in amphibians 3. In mammals it is believed to be involved in memory consolidation. Human patients with seizures or degenerations in the hippocampus have been reported to possess intact short-term and intact retrograde long-term memory, but they were found unable to permanently memorize new information24'26'43'53. Similarly, lesions or electrical stimulation through implanted electrodes in the hippocampal formation resulted in an inability of experimental animals to consolidate their longterm memory (e.g. ref. 16). Little is known, however, about the basic mechanisms involved in memory formation. Bliss and Lomo 6 discovered the phenomenon of long-term potentiation in the rabbit hippocampus which has been interpreted as an electrophysiological manifestation of memory formation 3°. Long-term potentiation of rat hippocampal slices has since been extensively studied as a model system for electrophysiological investigation of neural plasticity in vitro 22'54. Biochemical analysis of long-term potentiation s'9, like that of memory formation in general, is still at its very beginning. By means of radioactive double-labeling studies Duffy et al. 11 found that newly synthesized proteins were preferentially secreted from hippocampal slices during long-term potentiation. Recently Shashoua and collaborators reported the release of glycoproteins (molecular weights 86, 64, 32 and 14 kDa) and of S-100 from viable hippocampal slices in pulse chase and superfusion experiments 15'52. This finding at an electrophysiological model system for plasticity in the central nervous system bears some similarity to biochemical results obtained in connection with behavioral plasticity. It is

well known that memory consolidation implies protein biosynthesis and that two proteins specifically involved in long-term memory formation in goldfish, i.e. ependymins fl and 7 (32 and 26 kDa), are not only produced at a higher rate of synthesis45 but also secreted into the extracellular fluid when animals adopt a new pattern of swimming behavior 37'47. So far it was not known, however, whether the proteins synthesized during long-term potentiation in hippocampal slices (and possibly also in situ) are related to goldfish ependymins. By means of highly specific antisera against goldfish brain ependymins we have now been able to demonstrate ependymin-like immunoreactivity in rat brain hippocampus. Our results include several lines of evidence: (1) The antigenic material in the rat has been measured quantitatively with a very sensitive radioimmunoassay. (2) The highest values of crossreactivity were determined for the embryonic hippocampus, both in dissected brain and in neuronal cell cultures. (3) A cytoplasmic glycoprotein has been isolated from rat brain which comigrates with ependymin ), on SDS-PAGE and cross-reacts with it in the radioimmunoassay. (4) Various cell culture systems and cell type specific antisera have been used in an attempt to localize the immunoreactive substance to a specific cell population. (5) Clearly only neuronal cells derived from embryonic rat brain stained for ependymin-like immunoreactivity. The immunofluorescence was evenly distributed throughout the cell cytoplasm including the perikarya and all the dendritic extensions but was not detected in cell nuclei. (6) Fluorescence labeling was most prominent in a specific population of embryonic hippocampal neurons in culture which resemble pyramidal cells. Interestingly, other neuronal cell types derived from adult or newborn rats and mice, e.g. cerebellar neurons (unpublished results together with Dr. Althaus, G6ttingen, F.R.G.), did not reveal cross-reactivity with goldfish brain ependymins. It is not yet known, however, whether the ependymin-like immunoreactivity is characteristic of hippocampal neurons or whether it is typical for neurons of a certain developmental stage. The notion that little ependymin-like immunoreactivity is expressed in adult rat brain tissue (including the hippocampal formation) seems to indicate that only cells which are not fully differentiated, or 'plastic', or become so by dediffe-

256 rentiation in culture, can synthesize ependymins. It is remarkable in this context, that also neurons in the

ACKNOWLEDGEMENTS

optic tectum of goldfish where particularly high concentrations of e p e n d y m i n s have been measured 3-s un-

For generous gifts of their antisera we thank Dr. A. Bignami, Boston, U . S . A . ( a n t i - G F A P antise-

dergo mitosis and thus lack one characteristic of fully

rum), Dr. J. Cohen, L o n d o n , U.K. (anti-A4 anti-

differentiated n e u r o n s (cf. refs. 17 and 25). On the other hand, epigenetic plasticity of the central neurons during development and the behavioral plastici-

body), Dr.

ty during learning do not necessarily have any more in c o m m o n than the designation. Certainly this ques-

(anti-NSE antiserum), and Dr. B. Ranscht, L o n d o n , U.K. (anti-GalC antibody). We also thank A. Polot-

tion will have to await further elucidation.

zek for typing the manuscript, G. Meyer for excellent

K. Hallermayer, Wfirzburg, F . R . ( ; .

( a n t i - G F A P antiserum), Dr. P.G. Marangos, National Institute of Mental Health, Bethesda, U.S.A.

technical assistance and A. Heidt and H. Schneider for help with the photographic work. This work was supported by grants from the Deutsche Forschungsgemeinschaft (Schm 478/3 and 4 to R.S.).

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