Antibodies to ependymin block the sharpening of the regenerating retinotectal projection in goldfish

Brain Research, 446 (1988) 269-284 Elsevier

269

BRE 13449

Antibodies to ependymin block the sharpening of the regenerating retinotectal projection in goldfish John T. Schmidt 1 and Victor E. Shashoua 2 IDeparunent of Biological Sciences and Neurobiology Research Center, SUNY Albany, Albany, NY 12222 (U.S.A.) and 2Ralph Lowell Laboratories, Harvard Medical School, McLean Hospital, Belmont, MA 02178 (U.S.A. ) (Accepted 6 October 1987) Key words: Retinotectal prc~jection; Optic nerve regeneration; Ependymin; Radial gila; Synaptic stabilization; Minipump infusion; Goldfish

The regenerating optic nerve of goldfish first reestablishes a rough retinotopic map on the tectum, then goes through an activity dependent refinement that appears to involve the elimination of inappropriate branches from early regenerated arbors. Retinotopically appropriate branches and synapses may be stabilized because the normally correlated firing of neighboring ganglion cells could cause summation of their postsynaptic responses, making them more effective. Thus, refinement of the map may be similar in several ways to associative learning. In this study, we therefore tested whether ependymin, a major protein component of the extraceUular fluid that has been implicated in synaptic changes thou[;ht to be associated with learning a simple task in goldfish, may also be involved in refinement of the retinotopic map. Goldfish that had undergone unilateral optic nerve crush received intraventricular infusion of antiependymin lgG or of control IgG's beginning at 21 days postcrush. Tc.-tal recordings from these fish at 39-56 days postcrush showed that the projection had failed to sharpen, much as in the fish with activity blocked o~ synchronized; the average size of the multiunit receptive fields was 31° vs 11° normally. The field potentials elicited from these tecta by optic nerve shock were not significantly smaller than in controls, suggesting normal levels of synaptogenesis. Control projections, identically treated but infused with either unrelated IgG or Ringer's alone regenerated normally, giving multiunit receptive fields of 12°. Intact (non-regenerating) projections of the experimental fish were not rendered abnormal by the IgG treatment. Histology showed the retinas and tecta of the infused fish to be normal in appearance. The results show a specific block of sharpening by antiependymin IgG. The ependymal gila of the tectum stain positively for ependymin in normal fish. particularly the cell bodies in the ependymal layer. The tectum, particularly the ependymal layer, stains more intensely during regeneration, which appears to trigger increased synthesis of ependymins in the ependymal gila. This increase and the block of sharpening by specific antibodies to ependymin suggest a possible role for ependymin in activity dependent synaptic stabilization, possibly through its polymerization when calcium is focally depleted at active synapses.

INTRODUCTION Recent studies in visual d e v e l o p m e n t and regeneration indicate that m a n y fine details of circuitry may be established by the selective stabilization of appropriate connections from an initially m o r e diffuse set of connections 9"15"2t''35"39"45and that this selective stabilization requires normal patterns of visual activity. Features whose d e v e l o p m e n t is activity driven include the segregation of ocular d o m i n a n c e patches, both in developing m a m m a l i a n visual cortex 47 and in dually innervated tecta of fish and frog 421"27, the segregation of receptive field types in lateral geniculate

nucleus I and the r e f i n e m e n t of retinotopic maps both in lateral geniculate of cat ~ and in tectum of goldfish 7'22'31. In several cases, the emergence of precise connections appears to require the coincident activation of appropriate afferents to stabilize their inputs o n t o the c o m m o n postsynaptic cells 7"34"48 as the sharpening may be blocked by the synchronization of all inputs as well as by blocking activity in all afferents. Normally this coincident activation appears to derive from the correlated activity of neighboring ganglion cells of the same type, visually driven as well as spontaneous activity-" H~.54. This apparent r e q u i r e m e n t for coincident activa-

Correspondence: J.T. Schmidt. Dept. of Biological Sciences and Nt:urobiology Research Center, SUNY Albany, 1401)Washington Ave., Albany, NY 12222, U.S.A. 0006-8993/88/$03.50 ~ 1988 Elsevier Science Publishers B.V. (Biomedical Division)

270 tion of converging inputs bears a formal similarity to associative conditioning, one of the model systems for studies of learning and memory. The prevailing view of associative learning is that it involves a change in synaptic circuits, either an increased strength in transmission at existing synapses or an increased number of synapses from the specific inputs 5a8.4°'~'5~. Therefore, we were motivated to test whether ependymin, a protein component that is thought to be involved in the consolidation of memory in goldfish 37"41, might also be involved in the activity dependent sharpening of the retinotectal projection during the regeneration of the optic nelve in goldfish. Ependymin is synthesized by the ependymal gila and is continually released as a disulfide linked dimer of two polypeptide chains (37 and 31 kDa) into the brain extraceUular fluid (ECI:) 41. It was first identified by double labelling methods as a glycoprotein whose turnover rate increased in the brains of goldfish that learned a new pattern of swimming behavior 37"4°. Subsequent studies showed that injections of specific antibodies to ependymin into the fourth ventricle within 24 h after training could block subsequent recall 37"44,suggesting that the protein could be involved in some aspect of the consolidation process of long term memory formation. The protein has the caoacity to undergo irreversible polymerization in low calcium medium 4~. ~_ e sustained activity at convergent synapses can transiently lower calcium by as much as 70% at local sites in the ECF t6'2°, the resuiting deposition of polymerized ependymins as a matrix 43could play a role in promoting increased synaptic growth or elaboration at the sites of the participating inputs 4i. In this study, we have used osmotic minipumps to infuse antibodies to ependymin during the time of sharpening of the retinotopic projection following regeneration of the optic nerve in goldfish. The results show a specific block of sharpening by antiependymin but not by other antibodies, without retarding the reestablishment of synaptic transmission by the regenerating fibers. The antibodies also do not disrupt existing retinotopic connections in the non-regenerating projection. Furthermore, immunostaining was used to show the spread of the infused antibody in the tectum, to show the presence of ependymin in radial gila as well as ependymal gila of the tec-

tum, and to demonstrate a great increase in ependymin levels during regeneration. MATERIALS AND METHODS Common goldfish (13-16 cm in length) were purchased from Grassyforks Fisheries (Martinsville, IN, U.S.A.), kept at 20 °C, and fed every other day. Following anesthesia by immersion in a 0.1% solution of tricaine methanesulfonate (TMS, Crescent Res. Chem., Paradise Valley, AR, U.S.A.), the right op.. tic nerve was crushed in the orbit under visual control using fine jeweler's forceps as previously described 32. The fish were then revived. At 20-22 days postcrush, the fish were again anesthetized for implantation of the minipumps. The dorsal cranium was opened to expose the tecta. Micropipettes were used to microinject 15/~i of antibody solution through each rectum into the tectal ventricle below 31. For experimental fish, the antibody solution was 0.25 mg/ml of either of two polyclonal antibodies (isolated IgG fraction) h~ sterile Ringer's solution (microfiltered). These injections were made to achieve an immediate onset to the antibody's action. For controls, equivalent concentrations of antiS-100 or goat antirabbit IgG antibodies were employed. The antiS-100 was the kind gift of Dr. Blake Moore (Washington Univ., St. Leafs). The goat antirabbit was purchased from Cappel Labs, Pittsburgh, PA, U.S.A.). These same !gG solutions also filled the minipumps that were then attached to each fish's head by a cannula and infused their contents into the tectal ventricle. The caudal quarter of the tectal commissure was cut to allow the insertion of the tapered tip of the cannula into the third ventricle. This cannula was constructed from PE tubing drawn out to a tapered tip over a flame. It had two flanges that fit snugly on either side of the cranium to prevent any vertical motion of the shaft (Fig. 1). The assembly slid into a slit opening in the cranium made caudally from the main opening. Later the main opening was sealed with cyanoacrylate, and the cannula was also glued in place. Finally, a cap of dental acrylic was poured around the emerging cannula to hold it rigidly vertical. About 1 cm above the fish's head, an osmotic minipump (Model 2002, capacity 220/fl, Alzet Corp.,

271

OSMOTIC PUMP

:ANULA

~CRYLIC FLANGES ~CRANIAL ~OPENING

TECTUM ~

TECTUM

Fig. 1. Diagram of the surgical implantation of the cannula through the tectal commissure to dcliver the solutions to the third ventricle. The opening in the cranium is exaggerated to show a wider view of the brain. Note that the flanges around the cannula as it passes through the cranium prevent vertical motion, while the dental acrylic on top prevents pivoting around the point where it passes through the cranium.

Pain Alto, CA, U.S.A.) was inserted into the end of the cannula, and remained tethered to the fish's head as it swam. The pump was weighed before and after filling to determine the starting volume. After removal, the remaining volume was measured with a Hamilton microliter syringe and subtracted to give the volume delivered over the 2-5 weeks that it was in place. The pumps delivered an average of 2.6-4.4 /~l/day of the IgG solutions to the tectal ventricle (Table I). The fluid cavities in and around the brain total about 150-200 ~d so that a great deal of dilution of the antibody must occur. Tectal unit recordings. For recording, the fish were reanesthetized and the tectum was reexposed by cutting away the remaining dorsal cranial plate. Each

fish was placed in an eye-in-water apparatus designed so that the eye looks out onto a hemisphere for recording and mapping visual units as previously described 32. Briefly, the arbors of retinal ganglion cells were recorded with platinum tipped Wood's metal filled pipettes. Routinely, several units were recorded at each site, and the combined responsive area of the visual field was termed the multiunit receptive field. When these were roughly circular, the diameter was used as a measure of size; when they were oblong, the average of the long and short axes was used instead. Occasionally, single units could be discriminated by spike height so that their receptive fields could be mapped separately from the other units at that location. In all cases, magnification factors (in micrometers on the tectum per degree in thc visual field) for the two dimensions of the map were computed by linear regression as previously described 34. Field potentials. In several cases, fish were transferred from the unit recording apparatus to a second apparatus for recording the field potentials elicited by supramaximal stimulation of the optic nerve. These were recorded to assess the progress of synaptogenesis in the regenerating projections. The techniques were the ~ame as those of previous reports 3°" 33.36. Briefly, DC recordings were made with a Ringer's filled pipette from various depths in tectum relative to a distant ground electrode consisting of a chiorided silver wire. Recorded voltage traces were digitized, averaged (5 traces, 0.1 Hz repetition rate), stored and displayed using an LSI 11/23 microcomputer and a Tektronix 4662 digital plotter. Optic nerve shocks several times greater than maximal were used to insure activation of all of the fine regenerated optic fibers. Amplitudes of maximal negative responses recorded from the main retinal recipient lamina (superficial gray and white layer) were compared to those from a group of control regenerates from a previous study 33. hnnumohistochemistrv. Fish were anesthetized and perfused through the heart with cold Ringer's solution, followed by 2% paraformaldchyde in phosphate-buffered saline. The bra~.ns were removed and fixed for an additional hour in paraformaldehyde. They were then sunk overnight in 30% sucrose, embedded in OCT embedding medium for sectioning at 25/tin on a cryostat. The sections were mounted on

272 gel slides and air-dried before immunostaining. Sections were stained on the slide in a drop of antibody solution in a moist atmosphere, using the procedures recommended by the vectastain kit (Vector Labs., Burlingame, CA). The primary antibody (1:1000 or 1:2000 dilution) was always left on overnight. Also all sections were treated for a half hour in 2% H20 2 in methanol either before any other solution or after the primary antibody. A few cases were also treated with cold acetone for l0 rain before the primary antibody. The HRP reaction product was developed with diaminobenzidine as described in the vectastain kit. Controls included the omission of the primary antibody, the substitution of normal rabbit serum, and the prior immunoabsorption of the primary antibody with heat denatured extradural fluid from the cranium of the goldfish (known to be high in ependymin content). Antibody preparation. Polyclonal antibodies were raised in rabbits against electrophoretically pure ependymins as previously described 4°. Two antisera, ANNA and CHAMP with titers of 1 x 105 and 5 x 105 respectively in Western blot assays 52 were used to prepare the !gG by the ammonium sulfate precipita95 tion method-. This method eliminates all other protein and lipid components of the antisera. The IgG was reconstituted in phosphate-buffered saline at 0.9 and 1.0 mg/ml for A N N A and CHAMP respectively. Both A N N A and C H A M P were found to recognize only the polypeptide and not the carbohydrate epitopes of ependymin (unpublished data). This property is essential for our experiments since a recent study 42 indicates that ependymin contains a carbohydrate epitope, HNK-1, also carried by several neural cell adhesion molecules 6. Thus, one can only study ependymin participation in a given process by using IgG that exclusively reacts with its polypeptide epitopes. RESULTS

Electrophysiological studies Unit recordings and mapping. The infusion of antiependymin IgG into the tectal ventricles during regeneration of the retinotectal projection substantially prevented the sharpening of retinotopic precision. This result is based upon recordings from 7 fish that were infused with antiependymin from minipumps

DORSAL

J ~

z

E

~

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/ ..j~

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~-~"

2

C)1

.JL]Row c

/z

~,

r

MEDIAL

LATERAL

e c

21

i HP

lmm

5s v R

VENTRAL

Fig. 2. Map of a retinotectai projection regenerated during infusion of antiependymin antibody (CHAMP). The recording was made 55 days postcrush and one day after removal of the minipump (fish no. 5). At the right is a drawing of the tectal surface viewed from above. Caudomedial to the tectum is the point of entry of the cannula through the tectal commissure (dark area). Each point in the tectal array is an electrode penetration. The receptive fields recorded at these points, although enlarged, fall into an orderly array in the visual field similar to the array seen on the tectal surface and numbered accordingly. For clarity, 3 separate representations of the hemispherical visual field, one for each row of points A, B and C on the tectum, are presented. The contours within the visual field define the boundaries of the multiunit receptive fields. For convenience, the drawing of the tectum has been inverted about one axis so that tile arrays are oriented in the same direction: rostral on the tectum corresponds to nasal in the visual field, medial to dorsal, lateral to ventral, and caudal to temporal.

and 2 others that received injections only, compared with recordings from 5 fish infused with other antibodies or with Ringer's alone. In all experimental fish, the general effects on the map recorded from 39 to 56 days postcrush were the same. The overall organization of the retinotopic map was normal, but the multiunit receptive fields were greatly enlarged; that is, multiunit activity recorded at each tectal site could be driven from a much wider than normal area of visual field (Fig. 2). The organization of the retinotopic map or, the tectum is such that nasal visual field is represented on rostral tectum, temporal field on caudal tectum, dorsal field on medial tectum and ventral field on lateral tectum, which curves ventrally and is largely out of

273 view (Figs. 2-4). A precise retinotopic organization can be seen in maps of normal projections (Fig. 3), and a rough retinotopic organization can also be seen in the unsharpened maps of the experimental f;~h (Fig. 2). As the electrode was moved caudally from the rostral pole in systematic increments, the centers of the visually responsive areas in visual field were located progressively more caudally. The magnification factor (MF) that describes this relationship, expressed as the number of micrometers on the tectal surface corresponding to each degree in the visual field, can be calculated by linear regression for both the rostrocaudal and the mediolateral dimensions. These factors were not different for the experimental and control fish. In the rostrocaudal direction, the average MFs were 19.75 pm/° (1.32 S.E.M.) for the ependymin infused fish and 21.09/zm/° (1.48 S.E.M.) for the control infused fish. In the mediolateral direction, the MFs averaged 24.42/zm/° (1.38 S.E.M.) for the experimentals and 22.81/zm/° (1.96 S.E.M.) for the controls. These figures were not significantly different from each other or from those of normal fish of this size (20.2 and 24.7/tm/° respectively34). Thus, the reestablishment of a rough retinotopic map, that normally occurs by 35 days postcrush, was not prevented by the presence of the antiependymin IgG from 21 days postcrush when the fibers have just begun to form synapses 33. The maps from the antiependymin infused fish were, however, quite different from those of control regenerates identically infused with vehicle Ringer's solution and from uninfused control regenerates reDORSAL MEDZAL

o

c

LATERAL •

VENTRAL

.

c

~

r-

t mm

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NP 11t !t2

Fig. 3. Orderly map of a non-regenerating retinotectal projection recorded from a fish infused with antiependymin lgG (CHAMP, fish no. 1). On the opposite tectum of this fish, the regenerating projection was prevented from sharpening by the lgG (average multiunit receptive field size of 28.8°), but this non-regenerating projection remained retinotopically sharp. Conventions are as in Fig, 2.

DORSAL

--t

N

DrLATERAL 0 VENTRAL

F i g . 4.

Regenerated r e t i n o t e c t a l

C projection

21

I mm

40

HP R recorded

at 43

days postcrush in a fish infused with goat antirabbit lgG from 2] to 33 days (control fish no. 3). The map is retinotopic and most multiunit receptive fields are of normal size (10-13°), but two

are slightlylarger. This case was the least sharpened of the control fish. Conventionsare as in Fig. 2.

corded from 39 to 56 days postcrush. The recordings showed that the sizes of the multiunit receptive fields were consistently several times larger than controls in linear extent (compare Fig. 2 and Fig. 4, see also Table I). These results were similar to those obtained from regenerating projections whose activity was either blocked by tetrodotoxin (TI'X 32) or synchronized by stroboscopic illumination 34. Both of these treatments block the sharpening of the multiunit receptive fields. These fields were defined by the inclusive boundaries of the areas of the visual field from which visual stimuli drove activit,, at each tectal site. The enlarged multiunit recep,~ve fields appear to reflect abnormal convergence onto each tectal site of ganglion cell arbors from a wide area of retina. This was confirmed in the TTX and strobe experiments by recording single units both from the tectum and from the retina (ref. 30 and unpublished). In this study single units were also occasionally recorded from the tectal neuropil. These single units always had normal receptive fields for ganglion cells (6 cases, average of 11.6 ° vs 11.3 ° for normals32). The multiunit receptive fields were either roughly circular, or elliptical. Slightly more than half (54%) of the multiunit fields in the 9 experimental fish were elongated (by the criterion of a greater than 10% difference in the two major axes), and there was a very marked tendency for the elongation to be in the horizontal direction (approximately 70% of cases). The muitiunit fields of Fig. 2 are typical in being predominantly horizontally elongated, but several are larger than average, as this fish showed the strongest effect

274 TABLE I Multiunit receptive field sizes

MURF, multiunit receptive field; RF, receptive field; GAR, goat antirabbit (lgG). Fish/Ab

Conch.

(I) Experimental fish (1A) CHAMP Ix~ (1B) (1C) (non-regenerating side) (2A) CHAMP 1/4x (2B) (3) CHAMP Ix (4) ANNA l/4x (5) CHAMP 1/4x (6) ANNA 1/4x (7) ANNA l/4x (8) ANNA 1/4x (9) CHAMP 1/4x

Days of infusion

Flow rate (l~l/day)

22-36

3.7

22-36

3.0

22-36 22-47 21-54 21-41 21-36 21b 21,27,37 b

3.5 3.2 4.3 4.4 2.5 25 20,20, 10

Day of recording

n (R Fs)

MURF size (°) mean (S E. M. )

39 56 56 39 284 43 47 55 50 51 56 43

25 5 13 28 41 13 11 20 19 17 13 12

28.8 27.8 9.3 28.3 10.1 32.8 30.8 35.5 32.4 34.1 24.6 28.9

(1.6) (4.3) (0.4) (1.9) (1.9) (2.2) (1.8) (2.6) (2.4) (1.7) (1.2) (1.8)

Minipumpfish

1-7

21-41

3.5 (0.26)

47.5 (2.4)

17.3 (2.7)

31.8 (1.0)

(I1) Control fish (1) Ringer's (2) GAR (3) GAR (4) GAR (5) antiS100

l/4x 1/4x 1/4x 1/4x

21-39 21-33 21-33 21-34 21-36

4.7 4.'i 4.7 4.6 3.3

40 40 43 54 53

29 39 23 17 27

10.6 12.2 13.1 12.2 12.2

Avg. control

1/4x

21-35

4.4 (0.3)

46 (3.1)

27.0 (3.6)

12.1 (0.4)

(0.1) (0.5) (0.8) (0.4) (0.5)

a Full strength (1 x) was approximately 1 mg/mifor CHAMP and 0.9 mg/mlfor ANNA. b These two fish had injections only (no minipumps were attached) on the days listed, and in the amounts listed in the next column.

(Table I, fish no. 5). In the fish receiving control infusions, very few (less than 3%) of the much smaller multiunit receptive fields were elongated by this criterion, and the axes of elongation were roughly evenly distributed to horizontal, vertical and diagonal orientations. Thus, the multiunit receptive fields in the ependymin infused fish were not only greatly enlarged but also showed a distinct tendency to be elongated horizontally. The numerical dn.ta from the r~cordings ~re presented in Table I. The mean multiunit receptive field size for individual fish in the minipump infused experimental group ranged from 27.8 ° to 35.5 ° . This is significantly different both from normal regenerates 32"34and from the control fish of this study (t-test, P < 0.000!). There was no difference in the size of the effects produced by the two antibodies C H A M P and ANNA. Although the total time that the fish carried the minipumps varied from 14 to 33 days, this did not significantly affect the size of the multiunit receptive fields. The 4 fish that carried the pumps for the

shortest time (14 or 15 days), averaged 31.0 ° , while those carrying the pumps for the longest (20, 25 and 33 days) averaged 32.9 °. Likewise, a close analysis showed that the sizes were not correlated either with the flow rate from the pump, or with the total amount of antiependymin IgG infused. Thus, it would appear that sufficient IgG was infused even in the case with the slowest flow rate and shortest interval. Full strength or 1/4 strength solutions were both adequate, and we have not tried to determine what the maximum effective dilution would be. However, we have noted that sharpening could be largely blocked by a single injection of 15/~i administered into the tectal ventricle on day 21 without subsequent infusion (fish no. 8) or with 3 injections at 21, 27, and 36 days (fish no. 9). The infused or injected concentrations would be diluted substantially after they mixed with the 150-200 pl of intracranial fluids. For example, the daily infused volume from a minipump (3-4 /~!) would be diluted approximately 50-fold upon complete mixing with the intracranial fluids (proba-

275 bly more if there is an outflow of CSF from the cranial cavity). The control fish were injected and implanted with minipumps in exactly the same manner, and carried them for approximately the same lengths of time (Table I). The IgG concentrations of the goat antirabbit IgG and the antiS-100 IgG were the same as for the antiependymin, the infusion rates were very similar, and the fish were recorded at equivalent times postcrush (Table I). Neither of these antibodies nor the Ringer's alone had any substantial effects on the sharpening of the regenerated projections (Fig. 4). The average size of the multiunit receptive fields in these fish ranged from 10.6 to 13.1 °, and the average of 12.1° (0.4 S.E.M.) was not significantly different from previously recorded control regenerates (11.9 °, 0.4 S.E.M.34). Thus, there was no general effect either of the minipump implantation and infusion procedure or of non-specific IgGs on the sharpening of retinotopic precision. Thus, the highly significant effect (P < 0.0001) produced by the infusions may be attributed specifically to the antiependymin antibodies. In two cases we looked at the ability of the projection to recover after the infusion of antiependymin IgG was ended, that is, the capacity for delayed sharpening after the infusion was terminated. The first fish was recorded a second time at 56 days, or 17 days after the initial recording at 39 days, but little change had taken place during this interval. The average multiunit receptive field size was 27.8 ° at the second recording versus 28.8 ° initially. The second fish was rerecorded after a longer recovery period of more than 9 months. At 284 days, the average size of the multiunit receptive fields had dropped to 10.1° from an average of 28.3 ° at 39 days, and the projection had returned nearly to normal. However, in this projection there were 3 instances of double receptive fields in the map: instances where a single recording site has two spatially separate receptive fields. This abnormality was previously found in projections that had recovered from either strobe or TTX block of sharpening after many months 34. Thus, sharpening can take place slowly after the end of tile antibody infusion, as it does after discontinuation of strobe or TTX, but the resulting map is not always completely retinotopic. In each fish, only the right optic nerve was regen-

erating, while the projection of the left nerve remained intact. This intact projection was exposed t o the injected and infused antibody just as the regenerating one was. We were therefore interested in whether the retinotopic precision of intact projections would also be disrupted. When such a projection was recorded (Fig. 3), no abnormalities were found, and multiunit receptive fields were normal in size (Table I). Thus, only regenerating projections were sensitive to the presence of the antiependymin IgG. This also parallels observations previously made on disrupting sharpening with either strobe illumination or intraocular injection of TI'X 32-34. Field potential recordings. Because the unit recordings described above are thought to originate from the (presynaptic) terminal arbors of the optic fibers 32, the possibility presented itself that the antiependymin IgG might be preventing the formation of a retinotopically sharp projection by preventing or substantially retarding the formation of synapses, or blocking transmission, rather than interfering with the subsequent selective stabilization of retinotopic (and elimination of non-retinotopic) synapses. ~ine formation of regenerated synapses can be monitored in the return and growth of the postsynaptically generated field potentials elicited by supramaximal stimulation of the optic nerve 33. Since a previous study 33 had established the time course of the growth i/~ amplitude of the field potentials, we recorded field potentials in the antiependymin infused fish for comparison. An example of the field potentials is shown in Fig. 5. In 5 cases recorded at 43, 44, 50, 50 and 51 days postcrush, the field potentials were roughly normal both in laminar distribution (recordings made at 50/zm depth intervals throughout the tectal layers) and in amplitude for this stage of regeneration. The field potentials featured a prominent negative going wave in the superficial tectal lamina where the optic fibers are known to terminate, and a reversal to a positive going waveform in the deeper lamina, reflecting the vertical extracellular current dipoles set up in the vertically oriented dendrites by the activation of the superficial optic synapses 3°. The amplitude of the maximal negative component of the field potentials, which occurred at a depth of 150 gm, was measured for quantitative comparison. In the experimental fish, it ranged from 1.3 to 2.1 mV, averaging 1.52 mV (0.15 S.E.M.). The aver-

276

000 m 050 m

100 m 150 m 200 m 250~m

300/.Jm 350~m

400/Jm Fig. 5. Field potentials elicited by optic nerve stimulation recorded from the optic tectum 50 days postcrush in a fish infused with antiependymin lgG (ANNA) from day 21 to day 41 (fish no. 6). Recordings were made at 50 pm depth intervals from the surface to 450 pro, and the depths are shown to the left of each record, which is an average of 5 traces. The calibration pulse at the beginning of each trace is 1 mV in amplitude and 2 ms long. The traces are 80 r,,.: "~ng. Negative is downward in all traces. The optic nerve was stimulated at the small arrows, but the stimulus artifact was electronically suppressed. The amplitude of the maximal negative component in superficial tectum (150 pro) was 1.3 inV. Note the reversal to a positive going wave in deeper layers.

age amplitude in the control regenerates was 1.75 mV (0.29 S.E.M., n = 10). There was no significant difference between these two groups (P > 0.30, ttest; P > 0.40, Mann-Whitney U-test). In 4 of 5 cases, the field potentials were recorded after the unit recordings were made. These cases showed that these projections were unsharpened, but the unit recording procedure introduced a slight difference between the experimentals and the controls (in which unit responses were not first recorded) that might account for the slightly smaller potentials. Indeed, the fifth experimental fish, which had the field potentials

recorded immediately, showed the largest potentials (2.1 mV). In conclusion, these recordings, most of which were taken within 5-7 days after the end of the infusion, show no evidence that the antiependymin retards synaptogenesis. The field potentials were also examined for the latency of the responses following optic nerve shock, since this is an indication of conduction velocity which increases steadily as the regenerated fibers mature 33. The latency to the peak of the maximal negative component ranged from 31 to 43 ms, averaging 37.2 ms (2.01 S.E.M.). This was significantly longer than that of the control regenerates, which averaged 25.8 ms (1.35 S.E.M., n = 10) in both the ttest (P < 0.001, two-tailed) and the Mann-Whitney U-test (P < 0.002, two-tailed). This increased latency of the postsynaptic responses, provided there was no increase in synaptic delay, would indicate that the conduction velocity of the regenerating optic fibers was slower, possibly because the fibers were smaller in diameter or less myelinated. Longer than normal latencies were also found in the TTX blocked projections 33. Because the other treatments that prevent retinotopic sharpening interfere with normal visual activity in the pathway, we tested whether the antibody had any effect on synaptic transmission in a normal fish. Approximately 1 pl of a 0.25 mg/ml solution of the antiependymin IgG was slowly microinjected directly into the synaptic neuropil over 5 min. Recordings taken several minutes after the microinjection had maximal negative waves of 4.9 mV amplitude, almost the same as the 5.1 mV recordings obtained just before the microinjection. The field potentials after the injection did not differ in time course in any way from normal, either in latency, depths or duration. Thus the IgG itself did not appear to interfere with synaptic transmission.

Immunohistochemical studies In 6 cases, the brains from the minipump infused fish were examined anatomically. The sectioned material showed that the structure of the tectum was normal even around the cannula, and this material also localized, through the application of secondary antibody and avidin-biotin-peroxidase conjugate, the presence of the infused antiependymin IgG in the tissues.

277

Cytoarchitecture of infused tecta. Fig. 6 shows a camera lucida drawing of a cross-section through the tectum at the level of the entry of the cannula through the tectal commissure. Other than the hole in the commissure and slight accompanying gliosis around it, the cytoarchitecture of these brains was completely normal. Even the torus iongitudinalis, the small paired structures just medial to each tecturn, were normal in appearance except for a layer of gliosis above them. Localization of infused antiependymin IgG. In order to localize the infused antibody, the brains had to be lightly fixed since the antigenicity of the infused antibody was found to be much more sensitive to fixation than the antigenicity of ependymin itself (which would withstand 3 h fixation). However, a minimum of 1 h fixation was necessary to preserve the tissue structure during sectioning and staining. Therefore, the staining levels seen after treatment with secondary antibody and avidin-biotin conjugated peroxidase on the slide represent only a portion of the total antibody present (Fig. 6). The infused antibody was found to have bound within numerous anatomical structures in the brain. First, the cells of the gliosis around the entry zone were darkly stained, consistent with the gliai origin of this protein. Second, the staining in the tectum resembled that in normal tecturn but was more intense in some areas. There was a darker than normal staining of glial somata in the lower periventricular layer and in the ependymal zone (radial glia and ependymai cells respectively; nomenclature of Stevenson and Yoon~t'). There was, in addition, heavier than normal staining of cellular processes in the ependymal zone beneath the tectum and at the pial layer. These two zones (pia and ependymal zone) are the areas where the radial glial cells have their endfeet. The ependymal cells also contribute many processes to the ependymal layer a6. Finally, there was light to moderate staining of the neuropil of the tectum, including the optic recipient layers, but no staining of the neuronal somata (Figs. 6 and 7C~. This staining pattern was present with lesser intensity for more than a week after the infusion stopped. In the minipump infused fish, occasional radial gliai cells were stained in their entirety (Fig. 7C) probably because the prolonged presence of the antibody allowed penetration. The cell bodies, located in

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Fig. 6. A camera lucida drawing and two photomicrographs from a cross-section through the tectum at the point of entry of the cannula through the tectal commissure (ANNA infusion, fish no. 6). The section was treated with second antibody and ABC reagent to demonstrate the, presence of the antiependyrain IgG infused from the minipump. Shaded areas in the camera iucida drawing stained darkly for the [gG. The tecta and midline torus Iongitudinalis were undistorted in spite of the passage of the cannula through the commissure. The areas in the boxes are shown in the photomicrographs above and below. Note the stain in the gliosis (above) and in the ependymal and pial layers of the rectum (below). e, ependymai layer: pv, periventricular layer.

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Fig. 7. A collection of photomicrographs showing the immunostaining of ependymal and radial glial cells by antiependymin (A-C,F) and antiS100 lgG (E). A and B show weak staining of radial glia by ANNA in normal goldfish tectum (section treated on the slide with acetone and H202-methanol, photographic contrast enhanced by use of a deep blue filter). A: the ascending processes and pial endfeet. B: the cell bodies in deep periventricular layer and descending processes in ependymai layer. C: a single radial glial cell stained in its entirety by infused antiependymin (ANNA) in a minipump fish. D: a control section treated with normal rabbit serum instead of primary antibody. E: shows that antiS100 stains descending radial glial proce_sse~in the ependymal zone as well. Note the cell bodies, again in the lower level of the periventricular layer. F: the greatly increased staining with antiependymin (ANNA) found in the ependymal zone of tecta receiving regenerating projections (4 weeks postcrush). Staining with the CHAMP antibody produced nearly identical staining in all cases. Pv, periventricular cell layer.

the lower level of the periventricular layer, gave off both an ascending and a descending process. The descending process either ran all the way to the ventricle or to a blood vessel where it had an enlarged endfoot. The ascending process ran straight up, except for an occasional detour a r o u n d a large bundle of optic nerve fibers in stratum opticum, and ended in an

endfoot on the pia.

Staining in normal tectum. Radial glia were also found to stain faintly in normal brain sections that were treated on the slide with H 2 0 2 - m e t h a n o l and then with acetone. Fig. 7A,B, t a k e n with contrastenhancing blue filters, show that n u m e r o u s radial glia stain positively for e p e n d y m i n u n d e r these condi-

279 tions. The endfeet stood out at the pial surface and the radial processes ceuid be followed deep into the tectal neuropil and occasionally all the way to the cell bodies at the bottom of the periventricular layer (Fig. 7B). Cells with the same location and morphology as the radial glia also stain positively for S-100 (Fig. 7E), a cytoplasmic protein thought to be a good marker for glia49. Other cells staining darkly for ependymin, the ependymal glia, lie below the periventricular layer, and these are always seen with or without acetone treatment. The descending processes of these cells interdigitate with the descending fibers of the tectobulbar tract, oftentimes dividing them into large bundles (unstained areas, Fig. 6, bottom). Increased staining during regeneration. In those cases where the sections from the infused fish were retreated with antiependymin on the slide, it was obvious that there was a great increase in staining intensity over that in normal tecta. The neuropil itself stained moderately and the pial and ependymal zones were much more intense than normal. This increased staining suggested that synthesis of ependymin might be increased during the regeneration of the optic nerve. To test this possibility we stained tectal sections from control regenerates on the slide. The great increase in staining that occurs in the ependymal zone during regeneration can be seen by comparing Fig. 7F and B. The staining in this region changes from light staining of processes and cell bodies to a dark overall stain with still darker cells and processes often highlighted. This staining suggests an increased synthesis of ependymin triggered by the regeneration. DISCUSSION The main findings of this study are (1) that the infusion of antiependymin lgG into the tectal ventricles specifically blocks the sharpening of the retinotectal projection during regeneration; (2) that the infused IgG does not appear to hinder the reestablishment of synaptic transmission as assessed by the recording of field potentials elicited by optic nerve stimulation; (3) that immunostaining shows that the infused IgG had access to the tectal tissue; (4) that antiependymin stains some radial glia as well as the ependymal glia; and (5) that there is an apparent increase in cellular

ependymin staining during regeneration that suggests a possible increase in synthesis. Histochemical localization of ependymin. In goldfish brain, ependymin was previously localized using immunofluorescent staining to ependymai cells within the ependymal zones, such as that beneath the tecturn 3. Because ependymin is one of the most abundant proteins in the extracellular fluid 41 and because it turns over so quickly ~4"38,these cells that synthesize and release it are the most obvious structures stained in these sections. In both this and the previous study, there was light staining over the neuropil regions of tectum, and this may represent either polymerized ependymin or soluble ependymin that was fixed in place by the paraformaldehyde. The current studies also show staining i, cells of the ependymal zone, and in addition give more detail about their morphology, particularly the indication that some of them are in fact radial glia. The presence of the staining in the gliosis would appear to suggest that the astrocytes (often thought to be derivatives of radial glia) might also produce ependymin. Finally, the current study demonstrates a striking increase in ependymin staining levels during regeneration, which is consistent with the idea that it may play some role in regeneration. Whether this represents actual increased synthesis and the nature of the signal that might trigger the increased synthesis of ependymin remain to be investigated. Ependymin association with learning. A number of observations have implicated ependymin in synaptic changes associated with the consolidation process of long term memory formation. These include the observations that: (1) antiependymin antisera injected intraventricularly block the recall of long term but not short term memories after two different training paradigms in goldfish37"44; (2) immunocytochemical staining of brains of trained goldfish that could not recall the behavior because of intraventricular injections of the antibody showed the presence of discrete and s~ecific antibody stained sites in neuropil areas (at the light microscopic level) that were absent in the brain of antiependymin treated controls or trained animals that were injected with preimmune serum~: and (3) immunocytochemicai staining of potentiated rat hippocampal slices with antiependymin antisera and fluorescent second antibody showed the presence of dot-like fluorescently stained sites in stratum

280 radiatum, a region with large numbers of synapses 43. These experiments include both fish, where ependymins were first discovered, a,d mammals, where ependymi~.s are also present and secreted by certain a~troglia41. Antiependymin antibodies block sharpening. In these experiments, the infusion of antiependymin IgG blocked the sharpening of the regenerated retinotectal projection. The controls rule out the possibility that this effect could be due to any trauma associated either with the implantation of the cannula or with the infusion of the volume of Ringer's. The control antibody infusions also show that the effect is not produced by the presence of non-specific IgG's in the nervous system. The IgG's used were affinity pudried from two separate antisera and were monospecific for ependymin. First, they were raised by injecting electrophoretically purified ependymin into the rabbits, and second, their specificity was tested on Western blots where both antibodies recognized only the ependymin bands after electrophoretic separation of goldfish ECF and cytoplasmic proteins from tectum. Moreover, both ANNA and CHAMP antibodies were specifically directed towards polypepfide epitopes of ependymin, and had no detectable il~teraction with the carbohydrate epitopes of the molecule. The carbohydrate portion of ependymin has been recently found to stain with antibodies to HNK-1, an antigen which occurs within the carbohydrate w.oiety of certain neural cell adhesion molecules such as NCAM, J1, L1 and L2 (ref. 6). In spite of this, neither ANNA nor CHAMP recognized NCAM or other non-ependymin bands on Western blots of cytoplasmic or particulate fractions of goldu~h brain (Shashoua and Duffy, unpublished data). Goldfish NCAMs can be identified due to their recognition by antiNCAM directed against the polypeptide portion of mammalian NCAM, but the antiNCAM does not recognize the ependymins. Thus, the polypeptide portions of NCAM and ependymin are antigenically separate. Most importantly, although ependymin shares HNK-1 antigen with NCAM and other molecules, neither of our antibodies cross-react with it or other glycoproteins. Comparison with NCAM results in tectum. Fraser et al. 8 reported that the release of antiNCAM from small agarose pellets in Xenopus tectum could cause both a block of sharpening during regeneration of the

optic projection and also an unsharpening of a resident non-regenerating projection within a small area around the implant. Their results differ from ours in the latter respect, since antiependymin did not cause any disruption of the non-regenerating projection in our fish. That fact, combined with the lack of recognition of NCAM by these antibodies in Western blots, suggests that this result is produced by a different mechanism. Fraser et al. did not report whether mr not their antiNCAM treatment prevented the formation of normal levels of synapses or of synoptic transmission, but that would be important for defining the differences between the two effects, particularly since antibodies to NCAM have been reported to hinder the formation of neuromuscular synapses in vitro 28. NCAM is a cell surface glycoprotein, and the antiNCAM would presumably act by hindering cell-cell adhesion between presynaptic fibers and postsynaptic tectal cells. Ependymin, however, is an extraceUular glycoprotein, and because of its polymerization, apparently a matrix forming protein. Thus, a difference in mechanism should be expected. Soluble vs polymerizedforms. Given that there are soluble and polymerized forms of the ependymins, it is important to know which form the antibodies act against in preventing the sharpening. By calculating both the number of moles of antibody infused (assuming a molecular weight of 150,000) and also the number of moles of soluble ependymin in the extracellular fluids of the brain (assuming a minimum of 200 ~tl total of extradural, extracellular and cerebrospinal fluids and concentrations of at least 0.1% ependymin as measured by Shashoua41), we have calculated that there is more than 200 times more ependymin normally present than there is antibody in the initial injections. In addition, since ependymin has been shown to turn over with a half time of 2-4 h ~4'38 the amounts synthesized daily should exceed the antibody infused daily by even greater amounts. The soluble ependymin is largely in the form of fl-F dimers which are not very immunoreactive 4~, so that the infused and injected antibodies may diffuse freely through the fluids to bind to the polymerized form at specific sites. Shashoua 4~ previously demonstrated that these antibodies readily recognize the polymerized form. It is therefore likely that the antibody exerts its block of retinotopic sharpening by attaching to the polymerized matrix that may be deposited

281 at potentiated synapses 43. There, it might act to prevent growth of synapses over this matrix or cou~,d prevent further addition of polymerized deposits. The idea that extracellular matrix components may influenc~ the formation of synaptic structures is suppor(ed 0y observations on the neuromuscular junction where regenerating motor axons form presynaptic terminals at synaptic sites on empty basal laminae and where regenerating muscle fibers form postsynaptic specializations at such sites in the absence of the motor terminal. It appears unlikely that antiependymin interfered significantly with axonal growth and navigation since (1) visual units (optic terminals) could be recorded as usual by 39 days as in controls, and (2) the amplitude of the field potentials elicited by optic nerve stimulation was not significantly different from that of control regenerates. These field potentials, which invert polarity in deep layers, are a direct product of postsynaptic currents in the radially oriented dendrites of tectal cells3L33. Since such field potentials reflect currents generated by both large and small cells, they avoid possible problems of sample bias that may affect intraceilular recordings of EPSPs. In addition, where these potentials are small early in regeneration, their amplitude is likely to be a reasonable indication of the rough numbers of synapses formed. Likewise, antiependymin could not be acting to arrest regeneration at the stage at which it was first infused (21 days), since (i) retinal innervation does not cover all of the tectum at that time33; (2) no visual units can be recorded then32; and (3) the field potentials (that are present only in the extreme rostral tecturn) are extremely small (0.1 mV) in amplitude at that time 33. Thus regeneration appears to proceed in a substantially normal fashion in the presence of the antiependymin even though the map fails to sharpee. Thus, its interference seems to occur relatively late in regeneration, possibly with the retraction of inappropriate branches found on the initial optic arbors, or with the growth of additional retinotopically appropriate branches 35. Manipulations of activity that also prevent retinotopic sharpening (see below) appear to disrupt the selective eliminatioi~ or remodelling of branches resulting in abnormal regenerated arbors (Schmidt and Buzzard, unpublished data). Further experiments using anterograde HRP staining are planned to investigate the morphology of optic at-

bors regenerated in the presence of antiependymin IgG.

Parallels with the effects of activity manipulations. This study differs from other studies of retinotectal sharpening undertaken in the Schmidt lab 32"34 and elsewhere TM in that all previous manipulations that were found to prevent retinotopic sharpening during regeneration involved manipulations of activity. Here, the field potential experiments explicitly demonstrated that the antiependymin IgG did not affect synaptic transmission when microinjected directly into the neuropil. Thus, it does not affect activity. However, on a general level there are several parallels that have been noted between the effects of manipulating activity and those caused by infusions of antiependymin. First, a rough retinotopic map was formed in all cases and the multiunit receptive fields were enlarged to the same extent, indicating that sharpening was blocked to the same degree 32"3~. Second, in both types of treatment, the regenerating projection is prevented from sharpening, but resident non-regenerating projections are not unsharpened 32.34. Third, the regenerating projections that were initially kept from sharpening could later sharpen very slowly after the treatments were discontinued, resulting in maps with small multiunit receptive fields but with a few mistakes in the form of dual receptive fields for some tectal points 34. Finally, like both strobe (Eisele and Schmidt, unpublished) and TTX treatments 33, the infused IgG did not appear to hinder synaptogenesis, as assessed by the amplitude of the field potentials elicited by supramaximal stimulation of the optic nerve. One minor difference was that neither the intraocular TFX nor strobe illumination tended to produce elongated muitiunit receptive fields, that were seen in the most disrupted maps of this ~eries. The model of ependymin function. Very little is known on the biochemi, cal level about the processes controlling either synaptogenesis or subsequent synaptic stabilization. Previously, Shashoua 41 proposed a model of ependymin function based upon the fact that ependymin polymerizes in low calcium media, and may therefore be selectively deposited at highly active convergent synapses, since such synaptic activity has been shown to lower the levels of extraceilular c~icium 16'2°. In hippocampus. Shashoua and Hesse 4a showed that ependymin may become poly-

282 merized at potentiated synapses, and it might then serve as a favorable substrate for growth and elaboration of the synapses involved 41. This hypothesis becomes more attractive with the recent findings on the ionic channel properties of the N-methyl-o-aspartate (NMDA) receptor for excitatory amino acid neurotransmitters ~1,13,23,24. Excitatory amino acids rather than acetylcholine are now the suggested transmitter at the retinotectal synapses, since (1) choline acetyltransferase was not found in optic fibers 53, (2) many of the acetylcholine receptors were located on the presynaptic retinal terminals instead of postsynaptic to them 12 and (3) several general antagonists of excitatory amino acids were found to block retinotectal synaptic transmission 17. Whereas non-NMDA receptors (kainate and quisqualate receptors) open ion channels in the usual fashion, NMDA receptors have an additional valtage dependence tL~3, which gives them a conditional property 23, since they are blocked by magnesium ions except when the cell is already depolarized 24 by other simultaneously active inputs. When fully conducting, NMDA receptor channels readily pass calcium ions ~9, that serve as an intracellular messenger but which may also, by their depletion, cause extracellular changes. These properties explain how massive synchronous activity (such as during potentiation in hippocampus) can cause the observed drop in extracellular calcium, but they could also trigger smaller,

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