Journal of Neuroscience Research 33:626-630 (1992)

Biphasic Changes in NCAM Level After an NMDA Lesion to the Hippocampal Formation: A Quantitative Dot-Immunobinding Assay S. Wang, G.J. Lees, E. Bock, A. Hamberger, and K.G. Haglid Institute of Neurobiology, University of Goteborg, Goteborg, Sweden (S.W., A . H . , K.G.H.); Department of Psychiatry and Behavioural Science, University of Auckland School of Medicine, Auckland, New Zealand (G.J.L.); The Protein Laboratory, University of Copenhagen, Copenhagen, Denmark (E.B .)

With a quantitative dot-immunobinding assay, the time course changes of neuronal cell adhesion molecule (NCAM) concentrations and total tissue content were monitored in the rat hippocampus after a 40 nmol NMDA injection. A biphasic alteration was observed; a decrease occurred at day 3, an increase at day 30. The time course of changes differed from that of the glial fibrillary acidic protein (GFAP), a marker for reactive astroglial cell, but was similar to that for the markers of sprouting neurites, i.e., low (L) and high (H) molecular weight subunits of the neurofilament polypeptides. It is suggested that NCAM is implicated in the onset of neurite sprouting in the hippocampus after an excitotoxic trauma. 0 1992 Wiley-Liss, Inc.

Key words: NCAM, NMDA, rat hippocampus, dotimmunobinding assay INTRODUCTION Regenerative growth of neurites after a lesion is limited in the mature CNS (Chiquet, 1989). However, CNS neurons can regenerate into PNS grafts. Furthermore, regenerative responses are observed in situ in CNS neurons after a lesion (Cronin and Dudek, 1988). This correlates to collateral sprouting of intact axons and abortive sprouting of injured axons. We have shown that an N-methyl-DL-aspartic acid (NMDA) lesion to the hippocampus causes an initial decrease and a secondary increase of intermediary neurofilament polypeptides (NF-L and H) over 30 days (Wang et al., 1991). This result may reflect the outgrowth of neurites in surviving neurons in response to the damage. The neuronal cell adhesion molecules (NCAM), a family of membrane glycoproteins, have been implicated in supporting regenerative axonal growth in the PNS (Martini and Schachner, 1988; Rieger et al., 1988). In the CNS, NCAM is involved in the establishment and maintenance of functional cell-cell connections during 0 1992 Wiley-Liss, Inc.

development (Chuong and Edelman, 1984; Edelman, 1984; Silver and Rutishauer, 1984; Keilhauer et al., 1985; Pollerberg et al., 1985; Bhat and Silverberg, 1986; Beasley and Stallcup, 1987; Covault, 1989). There is little knowledge about whether NCAM is involved in outgrowth of neurites in adult mammals. We report here data showing temporal changes of NCAM following an NMDA lesion. Correlation analyses were carried out between NCAM and neurofilament proteins in the same brain area since the immunoreactivity of the neurofilament proteins has been found to increase when human and rat dorsal root ganglion neurons were cultured on monolayers of cells expressing transfected human NCAM (Doherty, 1990). The pattern of NCAM changes was similar to that of the L and H neurofilament proteins.

MATERIALS AND METHODS Animals Forty-four adult male Sprague-Dawley rats (200250 g) were randomly assigned to control or experimental groups. The latter were killed at various times after NMDA injection. The rats were kept four to six to a cage in a light-dark cycle (12 hr light/l2 hr dark) at a constant temperature of 22°C and 60% humidity and fed commercial laboratory rat chow (Astra/Ewos, Sodertalje, Sweden) and water ad lib. Injection Procedures The rats were anesthetized with sodium pentobarbital (60 mg/kg, i.p.) and positioned in a stereotaxic instrument with the nose bar set at -3.5 mm. The coorReceived May 28, 1992; revised August 3, 1992; accepted August 5, 1992. Address reprint requests to Prof. K.G. Haglid, Institute of Neurobiology, University of Goteborg, P.O.B. 33031, S-400 33 Goteborg, Sweden.

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Fig. 1. A standard curve of the dot-immunobinding assay for NCAM in SDS-sonicated rat brain homogenates ranging from 0.75 to 100 p,g tissue protein is presented. Control incubation was carried out by omitting the antiserum against NCAM from the procedure to check specificity of the assay. A background of 870 cpm has been subtracted from the values presented. Each point on the curve is the mean of eight measurements.

dinates of injection were: 4.4 mm posterior to bregma, 3.2 mm lateral to the central suture, and 2.5 mm ventral to dura. Forty nanomole NMDA in 1 pl 0.9% NaCl (neutralized to pH 6.5-7 with NaOH) was injected into the left hippocampus by means of a microinjection pump. Controls were injected with the same volume of 0.9% NaCl. The rate of injection was 0.5 pl/min and the cannula remained in place for an additional 5 min before withdrawal.

Preparation of Tissue Animals were decapitated after 3, 7, 10, or 30 days. Dorsal hippocampus (about A2.9-4.9 in the atlas of Konig and Klippel) was dissected out on ice, weighed, and frozen on dry ice. Tissues were homogenized by sonification (Branson, Sonifier, cell disrupter B 15) in 20 volumes of 1% sodium dodecyl sulphate (SDS) at 90°C until a clear solution was obtained (about 30 sec) and frozen at -20°C before analysis. NCAM Determination Samples were diluted in a sample buffer [ 120 mM KCl, 20 mM NaCl, 2 mM NaHCO,, 2 mM MgCl, 5 mM hepes, pH 7.4/0.7% Triton X-1OO(vol/vol)] to contain about 0.5 pg total protein per p1. Using the Minifold I1 slot-blot apparatus as a template, 20 pl of the diluted samples were blotted onto nitrocellulose (NC) membrane filters. The sheets were then fixed in 10%acetic acid and 25% 2-propanol. The remaining reactive sites were blocked in a blocking solution containing 0.5% gelatin. The sheets were then incubated overnight in an antibody solution (blocking solution, 0.1% Triton X-100, and sera). The antibody preparation (a mixture of antisera against NCAM-180, -140, and -120 kD) was diluted 1:

500. The specificities of the antisera have been described in detail (Linnemann and Bock, 1989). After antibody incubation, the sheets were reacted with '251-protein A in the blocking solution containing 0.1% Triton X- 100 at a radioactivity concentration in the range of 150,000200,000 c p d m l . Finally the sheets were dried, cut, and assayed for radioactivity in a gamma counter. The amount of NCAM in an unknown sample was quantified as follows: a reference sample was prepared from a mixture of all control samples in the frontal cerebral cortex. Standard curves were constructed from dilutions of this reference. The amounts of NCAM were determined by interpolation on the standard curves and expressed in arbitrary units (AU). One AU is the amount of NCAM in 20 pl of the reference sample. Protein concentrations in the samples were determined with a commercial assay (Pierce) in microtiter plates (Redinbaugh and Turley, 1986) using bovine serum albumin as the standard. Materials Nitrocellulose membranes (pore size = 0.45 pm) and a Minifold I1 slot-blot apparatus were obtained from Schleicher & Schuell (Keene, USA). '251-labelled protein A (7.71-8.31 pCi/pg; 1 Ci = 37 GBq) was from New England Nuclear (Dreieich, Germany). BCA Protein Assay Reagent was from Pierce (Rockford, USA). All other reagents were of analytical grade and were from commercial sources.

RESULTS Standard Curve and Precision of the NCAM Assay A typical standard curve in the dot-immunobinding assay for NCAM in rat brain homogenates is shown in

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Figure 1. The background was 870 t 44 cpm. The detection limit, i.e., the protein level in the homogenate with a total cpm equal to the cpm of the background plus two standard deviations, was 0.4 p g protein in whole brain homogenates. It was found that the change of precision, as shown by coefficients of variation, depended on the amount of homogenate applied to the NC membrane. Low and high amounts of sample proteins decreased the precision. This was previously observed in a similar assay procedure for other brain proteins (Wang et al., 1990). The coefficients of variation of the radioactivity recovered in homogenates containing 1.5 and 100 pg tissue protein were 9.3% and 14.4%, respectively. Application of a homogenate containing approximately 10 pg of tissue protein resulted in an intraassay variation of 4%. To check the specificity of the assay, control incubations were carried out by omitting the antiserum against NCAM from the procedure (Fig. 1). There was no unspecific binding of protein A to rat brain homogenates.

Determination of NCAM The NCAM concentration of the control was set to 100%. It was compared with that of NMDA injected animals. Saline injections per se have an effect on the level of certain proteins (Wang et al., 1991). NCAM is given both as concentration and as total amount per hippocampus. A biphasic alteration of the NCAM concentration was observed. There was a decrease at day 3 (30%) and an increase at day 30 (20%) (Fig. 2a). The total amount of NCAM in the NMDA injected dorsal hippocampus decreased markedly at 3 days with a significant recovery at 30 days (Fig. 2b). Correlation Analysis Between NCAM and Neurofilament Proteins A correlation analysis was carried out (Fig. 3) of the NCAM data and data of L and H polypeptides of neurofilaments in the same animals (Wang et al., 1991). There was a good correlation between NCAM and the two neurofilament proteins ( R = .703, P < .001 for NF68 and R = .738, P < .001 for NF200) after the NMDA lesion. DISCUSSION NCAM has previously been quantified with rocket immunoelectrophoresis (Bjerrum and Bog-Hansen, 1976) and competitive ELISA (Ibsen et al., 1983). This report shows that nitrocellulose membranes can be used as a solid support for the quantitative immunoassay of NCAM in hot SDS-sonicated rat brain extracts. SDS treatment reduces the standard deviation in the determinations (Brock and O’Callaghan, 1987). The results

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Fig. 2 . Percentage changes of N-CAM in the hippocampus following an NMDA lesion. The samples from the hippocampus ipsilateral to saline injection were used as the controls. Open and hatched bars represent the saline and the NMDAtreated samples, respectively. Standard deviations from four to six animals per group are shown. a: Arbitrary units/mg tissue. b: Total NCAM content in the dorsal hippocampus. Asterisks indicate a significant difference ( P < .05) from the control in the Student’s t-test.

were related to wet weight tissue but also expressed as total amount per brain structure since a brain edema would decrease the amount of per wet weight. Furthermore, extravasation of serum proteins produces a similar problem when the results are expressed on a total protein basis. Another problem is that a pronounced reduction of other proteins from the damaged area may increase falsely the amount of the marker of interest. The total amount of protein in a brain structure could not reflect a redistribution of cellular marker proteins. NCAM is relatively enriched in neurons (Linnemann and Bock, 1989) and its concentration should consequently be expected to reflect the time course of nerve cell destruction after a lesion. A delayed increase in concentration of three neuronal proteins, neuron specific endase (NSE), and the L and H form of the neurofilament triplet polypeptides, has been interpreted as a regeneration after a NMDA injection (Wang et al., 1991). The decrease in NCAM 3 days after the NMDA lesion could reflect the nerve cell loss. NCAM exists mainly in the membrane-bound form (98-99%) (Bock et al., 1987). This may explain why the magnitude of the decrease is smaller than that for NSE (Wang et al., 1991).

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The antibodies used in the study of Wang et al. (1991) were developed to detect newly synthesized NF-L and phosphorylated NF-H, the phosphorylation of which occurs when it is incorporated into intact filaments. The correlation between NCAM and the neurofilament proteins after a lesion provides support for their cooperative response. Finally, NCAM appears to be involved in sprouting of neurons after an excitotoxic event. One of the features of an excitatory amino acid lesion is the selective destruction of postsynaptic elements while presynaptic structures are protected (Oleny, 1978). There are three molecular forms of NCAM-120, 140, and 180 kD (Chuong et al., 1982). NCAM-120 is not detectable in synaptosomal membranes, whereas NCAM-140 is expressed on both pre- and postsynaptic membranes. NCAM- 180 is restricted to postsynaptic sites (Linneman and Bock, 1989). The present work may consequently be carried on further provided antibodies to these different NCAM forms are available.

ACKNOWLEDGMENTS

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REFERENCES

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Fig. 3 . Correlation analyses between NCAM and polypeptides of neurofilaments. a: NCAM and NF68. b: NCAM and NF200. The NCAM data of the NMDA treated animals were used for the analyses.

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