Neurochemical Research, Vol. 21, No. 5, 1996, pp. 585-593

Synaptic Degeneration and Remodelling after Fast Kindling of the Olfactory Bulb

David P. D. Woldbye , 1,3 Tom G. Bolwig, ~ Jorn Kragh, 1 and Ole Steen Jorgensen 2

(Accepted January 22, 1996)

Kindling of the olfactory bulb using a novel fast protocol (within 24 h) was studied in rats. In target brain regions, the effects of kindling were measured on the concentration of glial fibrillary acidic protein (GFAP) by dot-blot and on the concentrations of neural cell adhesion molecule (NCAM) and the 25 kDa synaptosomal associated protein of the D3 immunoprecipitate (D3(SNAP- 25)) by crossed immunoelectrophoresis. Bilateral increases in the levels of GFAP, indicating ac- tivation of astrocytes, were detected in primary olfactory cortical projection areas, including the piriform cortex, and also in the basolateral amygdala and dentate gyms, suggesting that these regions may be functionally altered during the kindling process. In the piriform cortex and dentate gyms increased NCAM/D3(SNAP-25) ratios found ipsilaterally at seven days after kindling prob- ably reflect an elevated rate of synaptic remodelling. At this time, however, an overall pattern of ipsilateral decreases in the synaptic marker proteins NCAM and D3(SNAP-25) indicated that this remodelling occurred on a background of synaptic degeneration. These results confirm previous studies showing that kindling is associated with synaptic remodelling and neuronal degeneration in the hippocampal formation and extends the area of plasticity to include the piriform cortex which is believed to be central to the kindling process.

KEY WORDS: Fast kindling seizures; synaptic remodelling, synaptic degeneration; astrocytic activation; GFAP; neural cell adhesion molecule; NCAM; SNAP-25.

INTRODUCTION

Kindling is the process whereby repeated applica- tion of an electrical or chemical stimulus, initially pro- ducing only weak seizure activity, progressively results in fully generalized motor seizures and induces a state of permanent, increased seizure susceptibility to the

Laboratory for Experimental Neuropsychiatry, Department of Psy- chiatry, The National University Hospital: Rigshospitalet-6234, Co- penhagen, Denmark.

2 Laboratory of Neuropsychiatry, Department of Pharmacology, Uni- versity of Copenhagen, Copenhagen, Denmark and Department of Psychiatry, The National University Hospital: Rigshospitalet-6102, Copenhagen, Denmark.

3 Address reprint requests to: David P. D. Woldbye, Laboratory for Experimental Neuropsychiatry, The National University Hospital: Rigshospitalet-6234, DK-2100 Copenhagen, Denmark.

585

stimulus. Kindling is recognized as a model of acute and chronic epileptogenesis (1). In addition, it has been sug- gested that a number of neuropsychiatric conditions might be explained in the context of a kindling-like mechanism (2,3). Several hypotheses have been pro- posed to account for the kindling phenomenon (4). In recent years, the "synaptic reorganization hypothesis" (5-7) has been receiving considerable attention. Accord- ing to this hypothesis, kindling causes sprouting with the formation of new synaptic connections which amplify recurrent excitation resulting in increased seizure sus- ceptibility.

Neural cell adhesion molecule (NCAM; 8-9), in the first studies termed "D2 protein" (10), is present in higher concentration in immature than in mature neu- ronal synapses and membranes (11,12). D3 is localized to the cytoplasmic leaflet of presynaptic plasma mem-

0364-3190/96/0500-0585509.50/0 �9 1996 Plenum Publishing Corporation

586 Wo|dbye, Bolwig, Kragh, and Jorgensen

branes and is par t icular ly enr iched in mature synapses

(11). The 25 kDa synaptosomal associated protein

(SNAP-25 (13)) is the immunoreac t ive componen t o f the

D3 precipi ta te in crossed immunoelec t rophores i s (14).

An increase in the calculated N C A M / D 3 ( S N A P - 2 5 ) ra-

tio reflects synaptic remode l l ing ( rev iewed in 12). By

compar ing the concentra t ion o f N C A M to that o f

D3(SNAP-25) , our group prev ious ly found ev idence o f

synaptic remode l l ing after seizures induced by electro-

convuls ive s t imulat ion (15-17) , but not after h ippocam-

pal k indl ing (18). It was hypothes ized that the lack o f

synaptic remode l l ing fo l lowing k indl ing as opposed to

E C S was due to the fact that r emode l l ing processes were

not coordinated because o f inter- individual differences

in k indl ing t ime (18). To further address this issue, it

was decided to study the N C A M / D 3 ( S N A P - 2 5 ) ratio us-

ing a protocol in wh ich kindl ing wou ld be comple t ed

wi th in 24 hours ( " f a s t k ind l ing" ) . Several fast protocols

for k indl ing have appeared (19-21) as al ternat ives to the

" t radi t ional k i n d l i n g " procedure o f one s t imulat ion per

day (22). These fast k indl ing studies used the h ippocam-

pal format ion as k indl ing focus. In the present study, the

k indl ing electrode was pos i t ioned in the ol factory bulb

because an effect o f the electrode i tself had p rev ious ly

been demonst ra ted in our b iochemica l test sys tem (23).

Moreover , the olfactory bulb has the advantage o f be ing

the mos t sensi t ive k indl ing site o f all s tudied regions,

requir ing the lowest number o f st imulations to kindle

(24). In addi t ion to N C A M and D3(SNAP-25) , glial fi-

br i l lary acidic protein (GFAP) was measured. G F A P is

an intermediate-f i lament protein expressed abundant ly

and a lmost exc lus ive ly in astrocytes (25). An increase in

G F A P is be l i eved to indicate " a c t i v a t i o n " o f astrocytes

and m a y reflect brain regions funct ional ly al tered during

the k indl ing process (1,26). Fur thermore , act ivated as-

t rocytes are important producers o f neurotrophic factors

which probably are direct ly invo lved in the control o f

synaptic r emode l l ing (27-28) .

EXPERIMENTAL PROCEDURE

A total of 54 male Wistar rats (MollegSrden, DK), weighing 290- 350 g at the time of surgery, were used. Under equithesin anaesthesia (SAD, DK; 3.3 ml/kg), concentric, bipolar, stainless steel electrodes (0.5 mm; Rhodes Medical, Ventura, USA) were chronically implanted into the fight olfactory bulb (6.1 mm rostral to bregma, 1.3 mm lateral to the midline, 2.5 mm ventral to the skull) in all animals. The incisor bar of the stereotaxic apparatus was adjusted to position bregma and lambda in the same horizontal plane. The electrodes were secured to the skull with seven jeweller's screws and a covering of dental cement and positioned into a headplug assembly as described previously (29). After a postoperative recovery period of 10 14 days, the animals were

randomly subjected to either fast kindling, sham-kindling, or tradi- tional kindling.

Fast Kindling. In pilot studies, the following procedure was found to establish kindling reliably within 24 hours. Kindling stimulation consisted of 2 s trains of 60 Hz, 1 ms, biphasic pulse waves. Seizures were rated according to the severity scale of Racine (30). A current of 150/xA peak-to-peak was applied every five minutes until a single grade 4 or 5 seizure or an aflerdischarge duration of at least 60 s was induced ("fast kindling criterion", FKC). If FKC was not achieved within 30 stimulations, the animal was excluded. Then followed a 90 min resting period. This procedure was repeated until a total of three grade 5 seizures were elicited ("fast-kindling end criterion", FKEC). Rats were also excluded if FKEC was not achieved within 24 hours. If at any time during kindling the afterdischarge duration was less than five seconds, the current of the following stimulation was increased by 50/zA. The room lighting was on during the entire kindling session, and the animals had flee access to food and water. Age-matched sham- kindling paired controls were handled in the same way, except for the passing of current.

Two, 7, 14, or 28 days after the beginning of the fast-kindling session, the permanence of the induced kindling response was tested. Kindling stimulations (same current as final value on the fast-kindling day) were applied with 5 min intervals until one grade 4 or 5 seizure was elicited or a maximum of 5 stimulations was delivered. Each sham rat received the same number of stimulations using the same current as its fast-kindled match. Immediately after these test stimulations, the brains of fast kindled and sham-kindled rats were quickly removed under halothane anaesthesia and kept at -30~ Six fast-kindled ani- mals were resubjected to the fast kindling procedure two or four days post-kindling (2- and 4-DPK). These were not analysed immunochem- ically.

Traditional Kindling. Traditional "slow" kindling was induced using the same kindling parameters as described above for fast kin- dling except that pulses were applied only once a day. Two, 7, 14, or 28 days after their third grade 5 seizure, the permanence of the induced kindling response was tested as described above. These animals were not analysed immunochemically.

tmmunochemical Analysis. Three coronal slices (2 mm thick) were cut from fleshly thawed brains using a rodent brain matrix (Ac- tivational Systems Inc., Warren, Michigan, USA). The caudal ends of the slices were located approximately at 0 mm (slice I), - 4 mm (slice II), and - 6 mm (slice III) with bregma as reference. The dissection was performed on ice, and the following structures were bilaterally excised: The olfactory tubercle from slice I, the dentate gyms, piriform cortex, basolateral amygdala (basotateral and basomedial nuclei), and perirhinal cortex (31) all from slice II, and the entorhinal cortex (lateral part) and substantia nigra (pars reticularis) from slice III. The concen- trations of NCAM and D3(SNAP-25) were measured in the regions as described previously (11,32). The tissues were homogenized by sonication in 4% Triton X-100 buffer at pH 8.6 to a final concentration of 3% to 4% (w/v). After solubilization for 20 h at 4~ the amounts of NCAM and D3(SNAP-25) were estimated by quantitative crossed immunoelectrophoresis using about 60-100/xg solubilized protein (es- timated by the Lowry method) precipitated with a polyvalent rabbit antiserum to rat brain synaptosomal membranes (11). GFAP was es- timated by dot-blot analysis of about 100 ng solubilized homogenate protein dotted on nitrocellulose (23). The GFAP in the dots was de- tected by incubation with a monospecific rabbit antiserum to GFAP (1:500; Dakopatts, Copenhagen, Denmark), followed by tz~I-labeled protein A (Amersham, Copenhagen, Denmark). The ~251 bound to the washed dots was estimated by scintillation counting. The specific con- centrations of each antigen were determined as the absolute concen-

Synaptic Degeneration and Remodelling after Kindling 587

Table I. Bilateral GFAP Changes in Target Regions at 2-DPK (n = 4-7), 7-DPK (n = 6-9), and 14-DPK (n = 4-6)

Brain region 2-DPK 7-DPK 14-DPK

Piriform cortex 72 (14) 6 85 (14) 6 23 (4) 6 80 (13) b 69 (14) 6 0 (2)

Entorhinal cortex 105 (7) 6 104 (7) 6 33 (10) 6 72 (10) 6 59 (11) 6 5 (5)

Perirhinal cortex 104 (19) 6 96 (13) 6 24 (9) 98 (21) 6 91 (12) 6 16 (5)

Basolateral amygdala 84 (34) 95 (22) 6 20 (5) 16 (21) 65 (19) 6 25 (5)

Dentate gyms 70 (6) 6 49 (17) 6 20 (6) " 76 (13) 6 49 (15) 6 18 (6)

Olfactory bulb 11 (1) 67 (11) 6 n.d. 4 (3) 19 (4) n.d.

Olfactory tubercle 17 (8) 44 (8) a 8 (8) n.d. 29 (25) -13 (8)

Substantia nigra 7 (21) -14 (5) -29 (9) 12 (16) 7 (8) -11 (10)

Values are percent change from sham (SEM). For each brain region the ipsilateral value is placed above the corresponding contralateral value; n.d. = not determined. ~ 6P<0.001, Bonferroni cor- rected t-test following significant ANOVA (kindled ipsi- or contralateral side vs. pooled sham).

tration (arbitrary units) relative to the total protein concentration. The specific concentrations were normalized and expressed as a percentage of the mean of the relevant tissue from sham rats. The marker proteins were measured separately in each brain region at each time point. However, in a group of sham rats, the marker proteins were measured simultaneously in all target tissues to allow for analysis of inter-re- gional variation.

Data Analysis. Immunochemical data were analysed using one- way ANOVAs followed by two-tailed t-tests according to the Bonfer- roni method with two pairwise comparisons (kindled ipsilateral or contralateral side vs. sham). Separate ANOVAs were performed for each marker protein in each brain region at each time point. Values of significance were used descriptively.

R E S U L T S

Fast Kindling. Approximate ly 85% of the rats sub- jected to the fast kindling protocol fulfilled the inclusion criteria stated above. The median total kindling time to reach FKEC was 16 h 52 min (minimum: 8 h 25 min; maximum: 21 h 40 rain). The median duration o f the total number o f recorded afterdischarges per fast-kindled rat was 20 min 37 sec (minimum: 11 min 33 sec; max- imum: 47 rain 52 sec). FKEC was achieved within a median o f 36 stimulations (minimum: 17; maximum: 67). Kruskal-Wall is one-way A N O V A by ranks revealed no significant differences between the four fast kindling groups neither regarding kindling time, afterdischarge duration nor the number o f stimulations to reach FKEC. In 70% of all the rats completing the fast kindling pro- tocol, the stimulation current did not have to be in-

creased above the start value o f 150 /zA. In the remaining rats, afterdischarges disappeared some time after the first grade 5 seizure, and the current had to be increased to 200 -450 /zA .

All rats at 7-DPK (n = 7), 14-DPK (n = 6), or 28- DPK (n = 4) developed grade 4 or 5 seizures on the first test stimulation. In contrast, no 2-DPK animals (n = 11) experienced more than grade 1 seizures within the five test stimulations.

When the fast kindling protocol was repeated ( " re - k indl ing") in a group of fast kindled rats at 2- or 4-DPK (n = 3, both groups), the afterdischarge threshold increased much faster than on the first kindling day. Thus, to induce the first FKC on the first kindling day, stimulation currents o f 150-200 /xA were used as op- posed to re-kindling values o f 500 /zA (2-DPK) and 350-450 /zA (4-DPK). Al l rats subjected to re-kindling at 2-DPK needed more than the 30 stimulations used as upper limit on the first kindling day for the induction o f the first FKC. However, with 30 additional stimulations following a 90 min resting period, the first FKC could be induced in all 2-DPK re-kindling animals within a range of 31-53 stimulations as opposed to the 5-11 needed in the same group on the first kindling day. Rats rekindled at 4-DPK were less inhibited and reached the first FKC within 11-15 stimulations as opposed to 7-9.

Traditional Kindling. All rats subjected to tradi- tional kindling developed the third grade 5 seizure within 7-11 stimulation days. Two, 7, 14, or 28 days later, one stimulation evoked a fourth grade 4 or 5 sei- zure in all rats.

Immunochemical Analysis. No significant side dif- ferences were found in any brain region o f the sham- kindled animals for either marker protein, not even in the olfactory bulb (electrode site). Consequently, ipsi- lateral and contralateral sham values were pooled and compared with ipsilateral and contralateral sides from kindled animals.

GFAP. Due to the high number of separate ANO- VAs performed in the GFAP analysis, a significance level o f p < 0.01 was used to avoid results o f spurious statistical significance. At all t ime points analyzed, ip- silateral GFAP concentrations in kindled animals were generally higher than the corresponding contralateral values. Compared to sham, GFAP increases were de- tected bilaterally in all brain regions of kindled rats at 2-DPK (Table I). Significant increases were found in the dentate gyms (70% and 76%, ipsilateral and contralat- eral to the kindling electrode, respectively), piriform (72% and 80%), entorhinal (105% and 72%), and per- irhinal (104% and 98%) cortices. GFAP remained sig- nificantly elevated in these structures at 7-DPK. In the

588 Woldbye, Bolwig, Kragh, and Jorgensen

225

= 200

,7s

~ , ~ 150 . ~

1oo

B LA ~ - - ~ 0 EC

"~'r h c ~ P i rC "x., OB

SN o 75

T-ql. 100 ' 15'0 ' 200 25'0

Basal levels of GFAP [ arbitrary units ]

'300

Fig. 1. The regional concentration of GFAP (arbitrary units) after kin- dling (7-DPK) plotted against the basal GFAP levels as measured con- tralaterally in sham-kindled rats. Values were fitted to a straight line. Linear regression values: y = -0 .6x + 266; p < 0.001, t = 6.00, df = 6; confidence interval (95%) of the slope = -0 .8 to -0 .3 . BLA basolateral amygdala, OB olfactory bulb, DG dentate gyms, EC en- torhinal cortex, PirC piriform cortex, PrhC perirhinal cortex, SN sub- stantia nigra, OT olfactory tubercle.

10 OB EC PirC BLA PrhC DG

-10

r *

0 , - 10 ~-~ D3 (SNAP-25)

e~ -20

�9 10 L

~ t W ' ' -10 OB EC PirC BLA PrhC DG

Fig. 2. The specific concentrations of NCAM, D3(SNAP-25), and the ratio of NCAM/D3(SNAP-25) expressed as the percent change from sham-kindled rats in six brain regions at 7-DPK (n = 6-9). Abbre- viations as in Fig. 1; error bars = SEM; open boxes around the bas- eline = SEM of sham; filled bars = ipsilateral side; striped bars = contralateral side. *p<0.05, **p<0.01, Bonferroni corrected t-test fol- lowing significant ANOVA.

piriform, entorhinal, and perirhinal cortices, the GFAP increases were of the same magnitude as that of 2-DPK. In the dentate gyms, however, the percent change over sham levels had decreased to 49% on both sides. In ad-

dition, at 7-DPK, significant increases were found bilat- erally in the basolateral amygdala (95% and 65%) and ipsilaterally in the olfactory tubercle (44%) and bulb (67%). GFAP had declined in all areas but remained elevated above sham levels in most regions at 14-DPK. Significant increases were still detected ipsilaterally in the dentate gyms (20%), piriform (23%) and entorhinal (33%) cortices. No significant changes were found in any region at 28-DPK (not shown). In the substantia nigra no significant changes were detected at any time point.

In sham-kindled controls, substantial region-to-re- gion differences were detected with regard to the specific concentration of GFAP (Fig. 1). Regression analysis re- vealed a significant inverse relationship between sham levels of GFAP and the kindling induced GFAP increase in a given brain region (p < 0.001). When the extreme value of substantia nigra was excluded, the linear rela- tionship remained significant (p < 0.01).

NCAM, D3(SNAP-25), and NCAM/D3(SNAP-25) Ratio. Changes in the specific concentration of NCAM and D3(SNAP-25) at 7-DPK are displayed in Fig. 2. Decreases in NCAM were noted ipsilaterally in all areas measured (the dentate gyms, olfactory bulb, entorhinal, perirhinal and piriform cortices) except the basolateral amygdala, but only reached statistical significance in the entorhinal cortex (10%). Generally, contralateral changes in NCAM were in the same direction but of smaller mag- nitude. All NCAM displayed the slower (adult type) of electrophoretic mobility and no evidence was seen of the faster moving polysialylated NCAM (NCAM-PSA (11, 12)). Likewise, decreases in D3(SNAP-25) were found ipsilaterally in all areas except the olfactory bulb. D3(SNAP-25) generally decreased more than NCAM. The D3(SNAP-25) decreases reached statistical signifi- cance in the dentate gyms (19%), entorhinal (15%) and piriform (12%) cortices. Contralaterally, changes in D3(SNAP-25) ranged from - 7 % to +3% without a con- sistent pattern.

The calculated NCAM/D3(SNAP-25) ratio was sig- nificantly elevated ipsilateratly in the dentate gyrus (15%) and piriform cortex (6%). Ipsilateral increases were also detected in the basolateral amygdala (8%), en- torhinal (4%) and perirhinal cortices (10%), but these were not significant. In the basolateral amygdala, how- ever, a high variance on the contralateral sides of kindled rats contributed severely to the total variance in the AN- OVA. Thus, when ipsilateral sides were compared to the sham group alone, NCAM/D3(SNAP-25) also increased significantly in the basolateral amygdala (p < 0.03, t- test). No changes were detected in NCAM, D3(SNAP- 25), or NCAM/D3(SNAP-25) at 14-DPK (not shown).

Synaptic Degeneration and Remodelling after Kindling 589

Inter-regional variations in the sham levels of NCAM, D3(SNAP-25), and NCAM/D3(SNAP-25) were also analysed (not shown). When specific concentrations or ratios in the analyzed brain areas were expressed rel- ative to the overall levels in whole cerebral cortex the most conspicuous region was the olfactory bulb with a relative NCAM/D3(SNAP-25) ratio of 2.0. In other regions the relative ratios were within the range of 0.7- 1.0. Regression analysis revealed no significant relation between sham levels of NCAM, D3(SNAP-25), or NCAM/D3(SNAP-25) ratios and the change following kindling. Furthermore, a regional kindling-associated in- crease in the NCAM/D3(SNAP-25) ratio or changes in NCAM and D3(SNAP-25) were not related to the in- crease in GFAP in the same region.

DISCUSSION

Fast Kindling. This paper describes a fast protocol for kindling (within 24 hours) of the olfactory bulb. Kin- dling is characterized by two basic criteria. First, the induction of generalized tonic-clonic seizures (grade 5 seizures) by repeated application of a stimulus which initially only produces electrographic seizure activity with no or mild motor manifestations; second, the per- manence of the kindled response. Thus after a stimula- tion pause of many days, the increased seizure susceptibility to the kindling stimulus persists. Since its first detailed description (22), kindling has been studied most frequently with stimulations applied once daily. This traditional procedure was shown to require the smallest number of stimulations (22). Hence, reducing the interstimulus interval to 30 rain, was found to in- crease the number of stimulations needed to kindle, while shortening the kindling time to about 11 h (33). Over the past ten years, however, fast kindling protocols have appeared using interstimulus intervals as short as 5 min (19-21). These are essentially comparable to tradi- tional kindling. The present protocol used 5 rain inter- stimulus intervals until FKC which was followed by a 90 min pause before the next series of stimulations. The pauses were introduced because pilot data indicated that stimulations following FKCs had lower efficacy with re- spect to elicitation of afterdischarges for approximately 90 min. Others have shown that motor seizures inhibit the elicitation of subsequent afterdischarges for 90 min (34). Even so, the present fast kindling protocol required almost four times as many stimulations as traditional ol- factory bulb kindling.

At 7-, 14-, and 28-DPK, the permanence of the pres- ent fast kindling protocol was comparable to traditional

kindling with one stimulation eliciting at least grade 4 sei- zures in all animals. In contrast, at 2-DPK, none of the rats experienced more than grade 1 seizures. When the entire fast kindling protocol was repeated at 2- or 4-DPK, the elicitation of motor seizures was clearly inhibited, and the AD threshold increased above that of the first kindling day. The activation of long-term inhibitory processes has been reported in other kindling studies which also used short interstimulus intervals (20,34,35).

GFAP. These data demonstrate that fast olfactory bulb kindling causes region-specific increases in GFAP. The increases were time-dependent, being maximal at 2- or 7-DPK depending on the brain region, and returned to baseline at 28-DPK. In most regions, GFAP increased bilaterally although ipsilateral effects were generally more pronounced. These findings are consistent with previous kindling studies (1,18,23,26,36,37).

It is generally assumed that a regional increase in GFAP is indicative of astrocytic activation. The revers- ible nature of the GFAP increase implies that this acti- vation per se is not likely to be causally related to the permanence of the kindling phenomenon. However, the astrocytic activation points to regions which are affected during kindling and may possibly be important to the kindling process. Consistent with this view, large GFAP increases were detected in the piriform cortex and ba- solateral amygdala both believed to be central to the kin- dling process (24,38). Large GFAP increases were also found in the dentate gyms and entorhinal cortex. The dentate gyms is an important control point to the spread of seizures into the hippocampus and possibly beyond (39). The entorhinal cortex is closely associated with the piriform cortex forming its caudal extension, provides an input to the dentate gyms, and receives direct projec- tions from the olfactory bulb (40). The large increases detected in the perirhinal cortex in the present paper sug- gest that this area may also be functionally altered by kindling. In support of this view, an altered relationship between the piriform cortex and perirhinal cortex has been reported after kindling (24). Thus, in kindled rats, perirhinal discharges are led by the piriform cortex but occur spontaneously in controls. Moreover, the perir- hinal cortex receives a direct bulbar input (40). In the olfactory tubercle, the increase in GFAP was small and only ipsilateral in spite of the fact that this region also receives direct projections from the olfactory bulb (40) and from most other olfactory structures (41). This might imply that this structure is not important to the kindling process. However, although an increase in GFAP certainly demonstrates that a brain area is affected by kindling, the proof for a direct role in the kindling process requires lesion studies.

590 Woldbye, Bolwig, Kragh, and J~rgensen

In the substantia nigra (pars reticularis), GFAP did not increase in accordance with previous studies (18,23). The substantia nigra, particularly the pars reticularis, has been claimed to be involved in seizure generalization (42,43). Prominent electrographic seizure activity occurs in the substantia nigra during kindled seizures (44) and 2-deoxyglucose autoradiography has revealed this area to be strongly metabolically activated during generalized kindled seizures (44,45). Like in a previous study (46), the highest basal level of GFAP were detected in the substantia nigra in the present study. The finding that an increase in GFAP is inversely related to pre-kindling GFAP levels (Fig. 1) might explain why kindled seizures do not appear to activate nigral astrocytes. They may already be maximally activated. Alternatively, nigral as- trocytes might provide efficient local protection against kindling effects which would otherwise increase GFAP. A protective function might also explain why the sub- stantia nigra is not a kindling site (22,42).

The fimctional implications of the GFAP increase (astrocytic activation) are unclear. GFAP is believed to be involved in the stability of astrocytic cell structure (25,47). Kindling-induced increases in GFAP are asso- ciated with hypertrophy of astrocytic processes and per- ikarya (1,26) suggesting that the GFAP increase reflects reorganization of the astrocytic cytoskeleton. Activated astrocytes promote neurite growth (48), and in the de- veloping brain astrocytes provide a scaffold for outgrow- ing axons guiding them to their destination (49). In this context, the increased GFAP might reflect an improved ability of astrocytes to structurally protect existing or outgrowing nerve fibres from possible kindling-induced damage.

The aspects of kindling which cause GFAP to in- crease in astrocytes remain to be established. Neuronal degeneration is a powerful signal for induction of GFAP as seen following lesions (27,50). Severe seizures cause neuronal injury (51). The overall pattern of decreases in NCAM and D3(SNAP-25) (see below) supports a con- tribution from neuronal degeneration in the present study. However, kindling-induced increases in GFAP and astrocytic hypertrophy have been reported without morphological signs of neuronal degeneration (36,37,52) suggesting that intense neuronal activity per se may also increase GFAP in astrocytes.

Finally, spreading depression has been reported to induce GFAP (53). There is evidence from pharmaco- logical kindling that epileptiform spikes trigger spread- ing depression in early kindling stages, while inhibiting it later in the kindling process (54). Spreading depres- sion accompanying electrically induced seizure activity may, in fact, account for a major proportion of the re-

sulting increase in GFAP (55). Thus spreading depres- sion might also contribute to the GFAP increases of the present study.

Synaptic Degeneration. The specific concentration of NCAM was significantly decreased in the ipsilateral entorhinal cortex of kindled rats. NCAM is a homophilic neural adhesion molecule which during brain develop- ment is expressed in both neurons and glial cells (9). In the adult brain it is mainly produced in neurons (56), although recent qualitative studies indicate that NCAM is transiently upregulated in activated hippocampal as- trocytes (57). In fetal neural cells a polysialylated form of NCAM (NCAM-PSA) predominates which is nearly absent in the adult brain. After lesions, re-expression of NCAM-PSA in the adult brain is found to be associated both with reactive astrocytes (58,59) and neurons (60). In the present study, we used crossed immunoelectro- phoresis to quantify NCAM. This method has the ad- vantage that the amount of both NCAM and NCAM-PSA can be estimated because the eleetrophor- etic mobility of NCAM-PSA is higher than the mobility of adult form of NCAM (11). However, we found no evidence of a significant amount of NCAM-PSA in kin- dled rats. Quantitatively, two membrane components are hypothesized to give the major contribution to the NCAM concentration in adult brain: mature synaptic membranes and the plasma membranes of fibers and synaptic membranes formed in the continuous process of synaptic remodelling (12). Therefore, in kindling the resulting concentration of NCAM is not only reflecting synaptic degeneration but is the net result of the com- bined decrease caused by synaptic degeneration and in- crease contributed by sprouting fibers and newly formed synapses (12). In contrast, the concentration of D3(SNAP-25) mainly reflects the density of mature pre- synaptic terminals, both during brain development and after brain lesions (11,12,61-63). D3(SNAP-25) is pres- ent in most synapses, regardless of their neurotransmitter specificity (13,64). After fast olfactory bulb kindling a significant loss of D3(SNAP-25) was found in ipsilateral dentate gyms, and entorhinal and piriform cortices, in- dicating loss of synapses in these brain areas. Previous studies, using stereological methods, have demonstrated neuronal loss in the dentate gyrus following three gen- eralized tonic-clonic kindled seizures (65,66). Further generalized seizure activity resulted in additional neu- ronal loss involving other limbic structures (65).

NCAM/D3(SNAP-25) Ratio and Synaptic Remod- elling. The localization of NCAM mainly to sprouting fibers and newly formed synapses and D3(SNAP-25) mainly to mature synapses suggests that the calculated NCAM/D3(SNAP-25) ratio reflects the amount of newly

Synaptic Degeneration and Remodelling after Kindling 591

formed neuronal processes relative to the amount of ma- ture synapses (12). Accordingly, an increased ratio is indicative of synaptic remodelling (12). A major meth- odologic advantage of the immunoelectrophoretic method used is that D3(SNAP-25) is precipitated along- side NCAM on the immunoelectrophoretic plate so that the concentration of NCAM in the brain sample can be directly compared with that of D3(SNAP-25). An in- crease in the NCAM/D3(SNAP-25) ratio has been shown to occur in a number of situations known or ex- pected to induce synaptic reorganization, including brain lesions (32), Alzheimer's disease (67), and electrocon- vulsive stimulation and learning (16,17). Hence, the present finding of significant increases in the calculated NCAM/D3(SNAP-25) ratio after fast olfactory bulb kin- dling in the dentate gyrus and piriform cortex provides evidence that kindling induces synaptic remodelling in these areas. Furthermore, increased NCAM/D3(SNAP- 25) ratios, albeit statistically non-significant, in the ba- solateral anaygdala, entorhinal and perirhinal cortices suggest that plastic synaptic changes may occur more widely in the limbic system during kindling. Several pre- vious kindling studies have demonstrated synaptic re- organization in the hippocampal formation. These have relied either on Timm staining to demonstrate aberrant mossy fibre sprouting in the dentate gyms (6,65) or on electron microscopic quantitation of hippocampal syn- aptic subtypes (52,68-71). To the best of our knowledge this is the first study to report evidence of synaptic re- modelling in the piriform cortex after kindling. The lack of NCAM/D3(SNAP-25) increases in a previous kin- dling study from our group (18) may have been due to inter-individual variation in the duration of the kindling process or to sampling differences. In the present study, the return to sham values of the NCAM/D3(SNAP-25) ratios at 14-DPK is probably best explained by the fact that newly-formed synapses have matured, and the syn- aptic remodelling process has terminated. The reason why, as shown in the present paper, increases in NCAM/D3(SNAP-25) were only found ipsilaterally in contrast to the bilateral GFAP increases is not clear. Pos- sibly, synaptic remodelling only occurs in the presence of neuronal degeneration which may predominantly be produced ipsilateral to the kindling electrode. In support of this view, the tendency of both NCAM and D3(SNAP-25) to decrease ipsilaterally (but not contra- laterally) in most sampled regions seems to indicate that kindling induces an ipsilateral nettodegeneration of neu- ronal processes.

In the dentate gyms, it has been proposed that the kindling-induced neuronal loss in the hilus followed by sprouting of mossy fibres leads to the establishment of

aberrant auto-feedback of the excitatory granule neurons (6,7). This would enhance the excitability of the granule neurons and could explain the increased seizure suscep- tibility of kindled animals. Sprouting in vitro has been shown to occur within hours (72). Mossy fibre sprouting is not observed until 2-3 weeks after the initiation of traditional kindling (6). Consequently, it has been argued that synaptic remodelling in the form of mossy fibre sprouting cannot account for the induction of kindling as seen with fast kindling protocols (21). However, strictly speaking the large number of kindling stimula- tions within a short time span of fast protocols might produce the sprouting response much faster.

A role for synaptic remodelling in the permanence of kindling is another possibility. This idea is supported by data from kainate epilepsy models showing that sprouting mossy fibres form functional feedback excita- tory connections (73,74). The sprouting correlates with the occurrence of chronic spontaneous seizures and EEG hyperexcitability (74). The reason why another study found no such correlation might be that kainic acid was administered subcutaneously (75) which damages other seizure sensitive structures than the dentate gyrus as op- posed to a supposedly more localized effect of intrahip- pocampal administration (74). Accordingly, the occur- rence of spontaneous seizures in rats without hippocampal sprouting after subcutaneous administration, might have been due to reported damage to the piriform cortex (75).

It remains uncertain, whether synaptic remodelling in the piriform cortex, as revealed in the present study, leads to the establishment of aberrant positive feedback projections resulting in increased seizure susceptibility. Such a role for synaptic remodelling in this area would be compatible with the piriform cortex as a central struc- ture to the kindling process (24).

In conclusion, our data support a role for synaptic remodelling in the kindling process.

ACKNOWLEDGMENTS

The technical assistance of Birgit H. Hansen and Gyda Centervall is greatly appreciated. This study was supported by the Danish Med- ical Research Council, Ivan Nielsens Foundation, Eli & Egon Larsens Foundation.

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