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Activity-dependent changes in synaptophysin immunoreactivity in hippocampus, piriform cortex, and entorhinal cortex of the rat
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Neuroscience Vol. 115, No. 4, pp. 1221^1229, 2002 A 2002 IBRO. Published by Elsevier Science Ltd All rights reserved. Printed in Great Britain 0306-4522 / 02 $22.00+0.00
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ACTIVITY-DEPENDENT CHANGES IN SYNAPTOPHYSIN IMMUNOREACTIVITY IN HIPPOCAMPUS, PIRIFORM CORTEX, AND ENTORHINAL CORTEX OF THE RAT S. LI,a;b I. REINPRECHT,c M. FAHNESTOCKb and R. J. RACINEa a b
Department of Psychology, McMaster University, 1280 Main Street West, Hamilton, ON, Canada L8S 4K1
Department of Psychiatry and Behavioral Neurosciences, McMaster University, Hamilton, ON, Canada L8N 3Z5 c
JSW-Research GmbH, Rankengasse 28, 8020 Graz, Austria
Abstract:Synaptophysin, an integral membrane glycoprotein of synaptic vesicles, has been widely used to investigate synaptogenesis in both animal models and human patients. Kindling is an experimental model of complex partial seizures with secondary generalization, and a useful model for studying activation-induced neural growth in adult systems. Many studies using Timm staining have shown that kindling promotes sprouting in the mossy ¢ber pathway of the dentate gyrus. In the present study, we used synaptophysin immunohistochemistry to demonstrate activation-induced neural sprouting in non-mossy ¢ber cortical pathways in the adult rat. We found a signi¢cant kindling-induced increase in synaptophysin immunoreactivity in the stratum radiatum of CA1 and stratum lucidum/radiatum of CA3, the hilus, the inner molecular layer of the dentate gyrus, and layer II/III of the piriform cortex, but no signi¢cant change in layer II/III of the entorhinal cortex, 4 weeks after the last kindling stimulation. We also found that synaptophysin immunoreactivity was lowest in CA3 near the hilus and increased with increasing distance from the hilus, a reverse pattern to that seen with Timm stains in stratum oriens following kindling. Furthermore, synaptophysin immunoreactivity was lowest in dorsal and greatest in ventral sections of both CA3 and dentate gyrus in both kindled and non-kindled animals. This demonstrates that di¡erent populations of sprouting axons are labeled by these two techniques, and suggests that activationinduced sprouting extends well beyond the hippocampal mossy ¢ber system. A 2002 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: axonal sprouting, immunohistochemistry, kindling.
widespread use of the Timm stain (Danscher, 1981; Danscher and Zimmer, 1978). The Timm stain has the major limitation that it labels only pathways containing heavy metals, most notably the mossy ¢ber pathway. Thus it is not clear whether there is activation-induced sprouting in other ¢ber tracts. Axonal sprouting can be measured more generally with biochemical markers of the nerve terminals such as synaptophysin (Masliah et al., 1990, 1993; Bergmann et al., 1997). Synaptophysin, which is a 38-kDa integral membrane glycoprotein, is a component of synaptic vesicles (Jahn et al., 1985; Wiedenmann and Franke, 1985; Sudhof et al., 1987). The precise function of synaptophysin is unclear, but it is involved in synaptic vesicle formation and exocytosis (Bergmann et al., 1993; Mundigl et al., 1993; Grabs et al., 1994; Papa et al., 1995; Papa and Segal, 1996). As a molecular marker for the presynaptic vesicle membrane, as well as a functional marker for synapses, synaptophysin has been widely used to investigate synaptogenesis in both animal models and human patients (Masliah et al., 1990, 1993; Alder et al., 1992; Mahata et al., 1992; Alford et al., 1994; Davies et al., 1998; Looney et al., 1999; Reinprecht et al., 1999; Hinz et al., 2001). Little is known about the relationship between the expression of synaptophysin immunoreactivity and electrical kindling, although synaptophysin immunohistochemistry
Sprouting of mossy ¢ber terminals is observed following seizures in human patients (Sutula et al., 1989; Ben-Ari and Represa, 1990; Houser, 1990) and in a variety of animal models including kainic acid- and pilocarpineinduced seizures (Longo and Mello, 1997; Ribak et al., 2000), head injury (Mathern et al., 1996; Golarai et al., 2001; Santhakumar et al., 2001), electroconvulsive shock (Gombos et al., 1999; Vaidya et al., 1999), long-term potentiation (Adams et al., 1997a), and kindling (Sutula et al., 1988; Represa et al., 1989, 1994; Stan¢eld, 1989; Sato et al., 1990; Represa and Ben-Ari, 1992; Rashid et al., 1995; Van der Zee et al., 1995; Golarai and Sutula, 1996; Watanabe et al., 1996; Adams et al., 1997b; Gombos et al., 1999). Kindling and long-term potentiation are used for studying activation-induced neural growth in adult systems, since these models can induce axonal sprouting in the absence of neural damage (Adams et al., 1997a, b). Axonal sprouting is most commonly studied in the mossy ¢ber pathway because of the
*Corresponding author. Tel. : +1-905-525-9140; fax: +1-905-5296225. E-mail address: [email protected] (R. J. Racine). Abbreviations : ANOVA, analysis of variance ; BSA, bovine serum albumin; IML, inner molecular layer ; PBS, phosphate-bu¡ered saline ; S.E.M., standard error of the mean. 1221
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has been used to demonstrate a connection between neural sprouting in the cortex and kainic acid-induced seizures (Chen et al., 1996) as well as in the hippocampus of epileptic patients (Davies et al., 1998; Proper et al., 2000). In this paper, we used synaptophysin immunohistochemistry to determine whether synaptogenesis can be induced in non-mossy ¢ber systems in the hippocampal formation and in the piriform and entorhinal cortices by electrical kindling.
EXPERIMENTAL PROCEDURES
Sections were then mounted on Superfrost-plus slides (Fisher Scienti¢c, USA) for immunohistochemistry as described previously (Reinprecht et al., 1999). In brief, mouse antiserum directed against rat synaptophysin (Boehringer Mannheim, ON, Canada) was diluted 1:50 in PBS containing 1.0% bovine serum albumin (BSA). Following blocking with 5% normal horse serum in PBS, slides were incubated with primary antibody overnight at 4‡C. Antigen/antibody complexes were visualized by the avidin^biotin^peroxidase complex technique (Hsu and Raine, 1981a; Hsu et al., 1981b) using biotinylated goat anti-mouse IgG (Vectastain, Vector Laboratories, CA, USA) diluted 1:100 in PBS^BSA and incubated for 30 min at room temperature, followed by 0.005% 3,3-diaminobenzidine-tetrahydrochloride (Vectastain, Vector Laboratories). Sections incubated without the primary antibody served as negative controls.
Animals
Densitometric analysis of synaptophysin immunoreactivity
Fourteen adult male Long^Evans hooded rats (Charles River, St. Constant, Quebec, Canada), weighing between 230 and 260 g at the time of surgery, were used in this study. All animal experiments were carried out in accordance with the Canadian Council on Animal Care Guide for the Care and Use of Laboratory Animals. E¡orts were made to minimize animal su¡ering and to minimize the number of animals used. Rats were housed individually, maintained on an ad libitum feeding schedule, and kept on a 12-h light/dark cycle.
To minimize the variability of synaptophysin-immunoreactive staining, sections from both groups of animals were stained simultaneously. Eight horizontal sections taken from each animal were examined at 50U magni¢cation using a Bioquant image analysis system (Bioquant-R and M Biometrics, Nashville, TN, USA) attached to a light microscope (Zeiss Axioskop, Oberkochen, Germany). To determine the density of the synaptophysinimmunoreactive label in the hippocampus, the optical densities of areas CA1 and CA3, dentate gyrus and background were measured by placing an open circular cursor with a diameter of 100 Wm at eight adjacent positions along the stratum radiatum of the area CA1 (Fig. 1A), 16 adjacent positions along the stratum lucidum/radiatum (starting at the hilus) of the area CA3 (Fig. 1B), and 10 adjacent positions along the hilus and inner molecular layer (IML) of the dentate gyrus, respectively (Fig. 1C). For the measurements taken from entorhinal and piriform cortices, three open circular cursors (diameter of 400 Wm) were placed along layer II/III of the entorhinal cortex (Fig. 1D) and three open rectangular cursors (1200 WmU300 Wm) were placed along layer II/III starting at the genu of the piriform cortex (Fig. 1E). For background staining, optical densities were measured within eight adjacent circular cursors of 100 Wm diameter placed along the corpus callosum. To control for variations in background synaptophysin-immunoreactive staining across sections, the average of eight background density readings from corpus callosum was subtracted from the density readings of each section. Analyses were done on both raw densitometry measurements and on measurements corrected for background density. Both the right and left sides of the brains were measured for all sections of each animal. In addition, to ensure that the optical density of synaptophysin immunohistochemical labeling accurately re£ects the quantity of synaptophysin-positive synapses, six adjacent circular cursors (diameter of 100 Wm) were placed across stratum lucidum/radiatum and molecular layers, perpendicular to CA3, and both the mean optical density of the synaptophysin-immunoreactive labeling and the number of synaptophysin-immunoreactive terminals were measured in the same six sections. The mean density values obtained were linearly correlated with the number of synaptophysin-immunoreactive sites (R2 = 0.9906, Fig. 2). The strength of the correlation suggests that the mean density measurements directly re£ect the quantity of synaptophysin-positive terminals.
Surgery and kindling procedures All rats were deeply anesthetized with sodium pentobarbital (65 mg/kg, i.p.) and treated with atropine sulfate (1 mg/kg, i.p.) to minimize tracheal secretions and stress responses. The animals were placed in a stereotaxic frame (David Kopf Instruments, Tujunga, CA, USA), and a bipolar electrode made from Te£on-coated, stainless steel wires (O.D. = 190 Wm) was implanted into the right perforant path (7.6 mm behind bregma; 4.1 mm lateral to the midline; 3.3 mm below the skull surface) (Paxinos and Watson, 1985). The electrode was held in place by dental cement and three stainless steel screws inserted into the skull. A fourth screw, with a male Amphenol pin attached, served as the ground electrode. Each animal was given 30 000 U of penicillin (i.m.) and oral Apo-sulfatrim following surgery. All animals were given 7 days for recovery from surgery before the initiation of the kindling protocol. Beginning on day 7 after surgery, the kindled rats received stimulations (twice daily for a total of 11 days) consisting of a 1-s train of 1-ms pulses at an intensity of 500^600 WA and a frequency of 60 Hz. This was su⁄cient to trigger an epileptiform afterdischarge after each stimulation, the duration and magnitude of which were determined from electroencephalographic recordings. Sham-stimulated animals were treated identically, but without any electrical stimulation. The behavioral progression of kindling-induced seizures was scored according to Racine’s standard classi¢cation (Racine, 1972): stage 1, mouth and facial twitches; stage 2, clonic head movements; stage 3, unilateral forelimb clonus followed by contralateral clonus; stage 4, clonic rearing; and stage 5, loss of postural control and falling. All seven kindled rats exhibited at least three consecutive stage 5 seizures by the end of the kindling procedure. Perfusion and synaptophysin immunohistochemistry Rats were deeply anesthetized with an overdose of urethane (1.25 mg/kg, i.p.) and perfused transcardially (descending aorta clamped) with 60 ml saline bu¡ered with 50 mM sodium phosphate (PBS), pH 7.4, followed by 200 ml 4% paraformaldehyde in PBS on day 46 postsurgery and/or on day 28 after the last kindling stimulation. The brains were removed, quickly frozen by immersing them in cold 2-methylbutane (Sigma) on dry ice and stored at 370‡C. After embedding the brains individually in OCT Tissue-Tek (Miles, Elkhart, IN, USA), horizontal, serial, 12-Wm sections were taken between 4.1 and 8.6 mm ventral to bregma (Paxinos and Watson, 1985) using a cryostat at 320‡C.
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Abbreviations used in the ¢gures DG ENT GC ML PC PIR SL SR
dentate gyrus entorhinal cortex granule cell layer molecular layer pyramidal cell layer piriform cortex stratum lucidum stratum radiatum
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Fig. 1. Digitized images of the hippocampal CA1 and CA3 ¢elds, dentate gyrus, and entorhinal and piriform cortices. Optical density measurements of synaptophysin immunoreactivity were performed by placing open circular cursors at eight adjacent postions along the stratum radiatum of CA1 (A), 16 adjacent positions along the stratum lucidum/radiatum of CA3 (starting from CA3c) (B), 10 adjacent positions along the IML and hilus of the dentate gyrus (C), three open circular cursors along layer II/III of the entorhinal cortex (D), and three open rectangular cursors along layer II/III starting at the genu of the piriform cortex (E), as described in the section on experimental procedures. DG, dentate gyrus; ENT, entorhinal cortex; PIR, piriform cortex. Scale bar = 100 Wm (A^D) and 300 Wm (E).
Data analysis
RESULTS
Four-way analysis of variance (ANOVA) (e.g. [4U(2U5U8)]) with one between variable (group) and three within variables [brain hemisphere (left or right), section (1^5 ventral to dorsal, in CA1, CA3, IML, and hilus; 1^3 ventral to dorsal, in piriform and entorhinal cortices), cursor position (1^8 in CA1; 1^16 in CA3; 1^10 in IML and hilus; 1^3 in piriform and entorhinal cortices)] was conducted for the analysis of synaptophysin immunoreactivity in the CA1, CA3, IML, hilus, piriform and entorhinal regions. The ANOVA was followed by post hoc Tukey comparisons. Data were considered signi¢cantly di¡erent at a minimum con¢dence level of P 6 0.05. All data reported in the present study are expressed as mean W standard error of the mean (S.E.M.).
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Synaptophysin immunoreactivity in CA1 and CA3 A four-way (groupU(brain hemisphereUlevelUcursor position)) ANOVA with one between and three within variables was conducted for the analysis of synaptophysin immunoreactivity in the stratum radiatum of CA1 and the stratum lucidum/radiatum of CA3. Analyses were done on measurements corrected for background density. Statistical analyses revealed no in£uence of seizure activity on the background staining in corpus callosum. The negative control slides were devoid of
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staining. The optical density of synaptophysin immunoreactivity in the CA1 ¢eld of the kindled animals was increased signi¢cantly, by approximately 48.9%, compared to the control animals [Fð1;12Þ = 18.25; P 6 0.005, Fig. 3A, B; Table 1]. In CA3, the density of synaptophysin immunoreactivity was also enhanced signi¢cantly, by approximately 51.5%, in the kindled groups relative to the non-kindled controls [Fð1;12Þ = 24.47; P 6 0.01, Figs. 3C, D and 4; Table 1]. Synaptophysin immunoreactivity was distributed in a gradient, with the optical density of synaptophysin immunoreactivity greatest in ventral and lowest in dorsal sections [Fð4;48Þ = 3.51; P 6 0.05, Fig. 5A]. The density of synaptophysin immunoreactivity in hilus and IML A three-way ANOVA (groupU(brain hemisphereU level)) was utilized to evaluate synaptophysin immunoreactivity in the hilus and IML of the dentate gyrus. There was a main e¡ect for group. Post hoc analysis revealed the synaptophysin immunoreactivity increased signi¢cantly, by 46.2%, in the hilus [Fð1;12Þ = 25.32, P 6 0.0005] and, by 56.9%, in the IML [Fð1;12Þ = 19.22, P 6 0.001] of kindled rats as compared with non-kindled rats (Fig. 3E, F; Table 1). In the IML, there was a main e¡ect for section. Post hoc Tukey tests showed that the optical density of synaptophysin immunoreactivity was greatest in ventral and lowest in dorsal sections of all animals [Fð4;48Þ = 3.32; P 6 0.05, Fig. 5B], which was similar to the distribution in the CA3 ¢eld. The density of synaptophysin immunoreactivity in piriform and entorhinal cortices A two-way (groupUbrain hemisphere) ANOVA was used to evaluate synaptophysin-immunoreactive presynaptic terminals in the piriform and entorhinal cortices. Post hoc Tukey tests showed an enhancement of 37.8% [Fð1;12Þ = 7.28, P 6 0.05] in the density of synaptophysin immunoreactivity in layer II/III in the piriform cortex of kindled rats as compared with controls (Fig. 3G, H; Table 1). However, there was no signi¢cant di¡erence in synaptophysin immunoreactivity in layer II/III in the entorhinal cortex between the kindled and non-kindled animals [Fð1;12Þ = 1.61, P s 0.05; Fig. 3I, J; Table 1].
Fig. 2. Plot of mean optical density of synaptophysin immunoreactivity (x-axis) vs. number of synaptophysin-immunoreactive sites (y-axis). These values were closely correlated in a linear manner.
DISCUSSION
The immunohistochemical labeling of synaptophysin provides a molecular marker for presynaptic vesicle membranes, allowing us to investigate the density of axon terminals in pathways that do not contain high levels of heavy metals, e.g. in the piriform and entorhinal cortices, as well as in previously studied hippocampal areas. In the present study, we demonstrate signi¢cant kindling-induced increases in synaptophysin immunoreactivity in the stratum radiatum of the CA1 and the stratum lucidum/radiatum of the CA3 ¢elds, the hilus, the IML of the dentate gyrus, and in layer II/III of the piriform cortex. No signi¢cant di¡erence was found in synaptophysin immunoreactivity in layer II/III of the entorhinal cortex of kindled rats compared to controls. We have demonstrated that the increased synaptophysinimmunoreactive optical density is directly correlated with the number of synaptophysin-positive terminals, suggesting that the kindling-induced increase in synaptophysin immunoreactivity re£ects sprouting of new terminals. However, we cannot rule out the possibility that kindling stimulation up-regulates synaptophysin protein in preexisting unlabeled terminals. Previous studies have reported no change in synaptophysin immunoreactivity following amygdala kindling (Hinz et al., 2001) or pentylenetetrazole kindling (Mahata et al., 1992). In those studies, synaptophysin immunohistochemistry was performed 2 days following the last stimulation. In the present study, we demonstrate that kindling increases synaptophysin immunoreactivity
Table 1. Mean density of synaptophysin immunoreactivity in hippocampus and cortices Group
n
Control
Kindled
CA1 CA3 Hilus IML Piriform (Wm32 ) Entorhinal (Wm32 )
7 7 7 7 7 7
18.62 W 0.81 18.53 W 0.79 23.94 W 1.27 19.58 W 1.60 0.037 W 0.00066 0.042 W 0.00063
27.73 W 1.60* 28.07 W 1.64* 35.01 W 2.74* 30.73 W 1.69* 0.051 W 0.00415* 0.043 W 0.00068
W S.E.M.; *P 6 0.05.
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Fig. 3. Representative synaptophysin-stained sections of CA1 (A, B), CA3 (C, D), dentate gyrus (E, F), piriform cortex (G, H), and entorhinal cortex (I, J). Sections from control rats are shown in A, C, E, G and I, and sections from kindled rats are shown in sections B, D, F, H and J. DG, dentate gyrus; ENT, entorhinal cortex; GC, granule cell layer; ML, molecular layer; PC, pyramidal cell layer ; PIR, piriform cortex; SL, stratum lucidum ; SR, stratum radiatum. Scale bar = 100 Wm.
in both the hippocampal formation and the piriform cortex 28 days following the last kindling stimulation. Mossy ¢ber sprouting is not detectable until at least 4 days following stimulation and is detectable over a period of months (Cavazos et al., 1991). Therefore, it is likely that previous studies failed to detect changes in synaptophysin-immunoreactive protein because sprouting is not yet evident 2 days after activation.
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Patterns of synaptophysin immunoreactivity compared to Timm stain The terminals of the mossy ¢ber axons that originate from granule cells of the dentate gyrus contain high levels of zinc, making the Timm stain an excellent means to track changes in the mossy ¢ber system. However, this technique is not e¡ective for monitoring axonal sprout-
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Fig. 3 (G^J).
ing outside the mossy ¢ber system. Our previous studies showed that Timm granule density increased markedly in the stratum oriens of CA3 and in the IML of the dentate gyrus of kindled rats compared to controls (Rashid et al., 1995; Van der Zee et al., 1995; Adams et al., 1997a,b). In the present study, we demonstrate that the optical density of synaptophysin immunoreactivity is also increased signi¢cantly in the stratum radiatum of CA1 and the stratum lucidum/radiatum of CA3, as well as in the hilus and IML of the dentate gyrus in kindled animals. Synaptic reorganization of the mossy ¢ber pathway following seizures in kindled animals is often associated with the development, progression, and permanence of kindling-induced seizures (Sutula et al., 1988, 1992; Cavazos et al., 1991; Dudek et al., 1994; Represa et al., 1994). Our experimental results indicate that kindling-induced seizure activity is also correlated with sprouting in other hippocampal pathways as measured by an independent molecular marker. While Timm granule density is greatest in the stratum oriens of CA3 near the hilus and decreases with increasing distance from the hilus (Rashid et al., 1995; Van der Zee et al., 1995; Adams et al., 1997b), synaptophysin immunoreactivity is lowest in the stratum lucidum/radiatum of CA3 near the hilus and increases with increasing distance from the hilus in kindled animals. Also, Timm granule density in CA3 is reliably greater in dorsal com-
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pared to ventral sections (Van der Zee et al., 1995; Adams et al., 1997b), whereas synaptophysin immunoreactivity is greatest in ventral and lowest in dorsal sections. The graded distribution of synaptophysin immunoreactivity in IML of the dentate gyrus is similar to that in the stratum lucidum/radiatum of area CA3. The di¡erent characteristics of synaptophysin immunoreactivity and Timm labeling in CA3 likely re£ects the labeling of di¡erent ¢ber and cell types within di¡erent layers or locations of hippocampus.
Fig. 4. Mean synaptophysin-immunoreactive optical density in the stratum lucidum/radiatum of CA3. Synaptophysin immunoreactivity was enhanced signi¢cantly in the kindled group (n = 7) relative to the non-kindled control group (P 6 0.01, n = 7). Error bars represent S.E.M.
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interictal spikes in the kindling model (Racine et al., 1988; McIntyre and Plant, 1989; McIntyre and Kelly, 1990; Stripling and Patneau, 1990; Haberly and Sutula, 1992; Teskey and Racine, 1993), but also one of the fastest brain sites for the progression of electrical kindling (Goddard et al., 1969; Racine, 1975; Cain, 1977). Recent studies with kindling and convulsant drug models of epilepsy suggest that the piriform cortex may be particularly susceptible to generation of epileptiform activity (Loscher and Ebert, 1996; Timofeeva and Peterson, 1997, 1999; Potschka et al., 2000). Kindling causes a marked and spontaneous ¢ring in neurons located at the transition between piriform cortex layers II and III (Gernert et al., 2000). Our ¢nding that electrical kindling up-regulates synaptophysin immunoreactivity in piriform cortex, but not in entorhinal cortex, is consistent with these results. It remains to be determined whether synaptic reorganization plays a critical role in the piriform cortex’s contribution to epileptogenesis. Conclusion
Fig. 5. Graded distribution of synaptophysin immunoreactivity according to section in kindled and non-kindled animals. (A) CA3, (B) IML. Synaptophysin immunoreactivity was greatest in ventral and lowest in dorsal sections (P 6 0.05, n = 14). Error bars represent S.E.M.
Synaptic sprouting in the piriform cortex and the role of the piriform cortex in kindling We ¢nd that the average terminal density as measured by synaptophysin immunoreactivity in layer II/III of piriform cortex is increased signi¢cantly in kindled rats, whereas no signi¢cant di¡erence is found in the entorhinal cortex between kindled and control rats. The piriform cortex is reportedly not only a primary locus for the generation of spontaneous, epileptiform,
We have found that kindling up-regulates synaptophysin immunoreactivity in the stratum radiatum of area CA1, the stratum lucidum/radiatum of area CA3, hilus, IML of the dentate gyrus, and piriform cortex 28 days following stimulation, but has no signi¢cant e¡ects in entorhinal cortex of the adult rat. It appears that seizure-induced synaptic reorganization extends well beyond the hippocampal mossy ¢ber sprouting shown with Timm stain. The piriform cortex, in particular, is an active site according to synaptophysin-immunoreactive staining. Even in the termination ¢elds of the mossy ¢bers, synaptophysin immunoreactivity revealed di¡erent gradients than were found with Timm labeling. This suggests that di¡erent populations of sprouting axons are labeled by the two techniques. Acknowledgements+This work was supported by Grant # MOP-13248 from the Canadian Institutes of Health Research to M.F. and R.J.R. I.R. was supported by a scholarship of the O⁄ce of International Relations of the Karl-Franzens University, Graz, Austria.
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Activity-dependent changes of the presynaptic synaptophysin-synaptobrevin complex in adult rat brain. Eur. J. Cell Biol. 80, 615^619. Houser, C.R., 1990. Granule cell dispersion in the dentate gyrus of humans with temporal lobe epilepsy. Brain Res. 535, 195^204. Hsu, S.M., Raine, L., 1981a. Protein A, avidin, and biotin in immunohistochemistry. J. Histochem. Cytochem. 29, 1349^1353. Hsu, S.M., Raine, L., Fanger, H., 1981b. The use of antiavidin antibody and avidin-biotin-peroxidase complex in immunoperoxidase technics. Am. J. Clin. Pathol. 75, 816^821. Jahn, R., Schiebler, W., Ouimet, C., Greengard, P., 1985. A 38,000-dalton membrane protein (p38) present in synaptic vesicles. Proc. Natl. Acad. Sci. USA 82, 4137^4141. Longo, B.M., Mello, L.E., 1997. Blockade of pilocarpine- or kainate-induced mossy ¢ber sprouting by cycloheximide does not prevent subsequent epileptogenesis in rats. Neurosci. Lett. 226, 163^166. Looney, M.R., Dohan, F.C., Jr., Davies, K.G., Seidenberg, M., Hermann, B.P., Schweitzer, J.B., 1999. Synaptophysin immunoreactivity in temporal lobe epilepsy-associated hippocampal sclerosis. Acta Neuropathol. (Berl.) 98, 179^185. Loscher, W., Ebert, U., 1996. The role of the piriform cortex in kindling. Prog. Neurobiol. 50, 427^481. Mahata, S.K., Marksteiner, J., Sperk, G., Mahata, M., Gruber, B., Fischer-Colbrie, R., Winkler, H., 1992. Temporal lobe epilepsy of the rat: di¡erential expression of mRNAs of chromogranin B, secretogranin II, synaptin/synaptophysin and p65 in sub¢eld of the hippocampus. Brain Res. Mol. Brain Res. 16, 1^12. Masliah, E., Mallory, M., Hansen, L., DeTeresa, R., Terry, R.D., 1993. Quantitative synaptic alterations in the human neocortex during normal aging. Neurology 43, 192^197. Masliah, E., Terry, R.D., Alford, M., DeTeresa, R., 1990. Quantitative immunohistochemistry of synaptophysin in human neocortex: an alternative method to estimate density of presynaptic terminals in para⁄n sections. J. Histochem. Cytochem. 38, 837^844. Mathern, G.W., Babb, T.L., Leite, J.P., Pretorius, K., Yeoman, K.M., Kuhlman, P.A., 1996. The pathogenic and progressive features of chronic human hippocampal epilepsy. Epilepsy Res. 26, 51^61. McIntyre, D.C., Kelly, M.E., 1990. Is the pyriform cortex important for limbic kindling ? In: Wada, J.A. (Ed.), Kindling, Vol. 4. Plenum, New York, pp. 21^32. McIntyre, D.C., Plant, J.R., 1989. Pyriform cortex involvement in kindling. Neurosci. Biobehav. Rev. 13, 277^280. Mundigl, O., Matteoli, M., Daniell, L., Thomas-Reetz, A., Metcalf, A., Jahn, R., De Camilli, P., 1993. Synaptic vesicle proteins and early endosomes in cultured hippocampal neurons: di¡erential e¡ects of Brefeldin A in axon and dendrites. J. Cell Biol. 122, 1207^1221. Papa, M., Bundman, M.C., Greenberger, V., Segal, M., 1995. Morphological analysis of dendritic spine development in primary cultures of hippocampal neurons. J. Neurosci. 15, 1^11. Papa, M., Segal, M., 1996. Morphological plasticity in dendritic spines of cultured hippocampal neurons. Neuroscience 71, 1005^1011. Paxinos, G., Watson, C., 1985. The Rat Brain in Stereotaxic Coordinates, 2nd edn. Academic Press, Sydney. Potschka, H., Schwabe, K., Loscher, W., 2000. Development of kindling and spontaneous seizures after massed stimulation of di¡erent loci in the rat piriform cortex. Brain Res. 855, 252^259. Proper, E.A., Oestreicher, A.B., Jansen, G.H., Veelen, C.W., van Rijen, P.C., Gispen, W.H., de Graan, P.N., 2000. Immunohistochemical characterization of mossy ¢bre sprouting in the hippocampus of patients with pharmaco-resistant temporal lobe epilepsy. Brain 123, 19^30. Racine, R.J., 1972. Modi¢cation of seizure activity by electrical stimulation. II. Motor seizure. Electroenceph. Clin. Neurophysiol. 32, 281^294. Racine, R.J., 1975. Modi¢cation of seizure activity by electrical stimulation : cortical areas. EEG Clin. Neurophysiol. 38, 1^12. Racine, R.J., Mosher, M., Kairiss, E.W., 1988. The role of the pyriform cortex in the generation of interictal spikes in the kindled preparation. Brain Res. 454, 251^263. Rashid, K., Van der Zee, C.E.E.M., Ross, G.M., Chapman, C.A., Stanisz, J., Riopelle, R.J., Racine, R.J., Fahnestock, M., 1995. A nerve growth factor peptide retards seizure development and inhibits neuronal sprouting in a rat model of epilepsy. Proc. Natl. Acad. Sci. USA 92, 9495^ 9499. Reinprecht, I., Gschanes, A., Windisch, M., Fachbach, G., 1999. Two peptidergic drugs increase the synaptophysin immunoreactivity in brains of 24-month-old rats. Histochem. J. 31, 395^401. Represa, A., Ben-Ari, Y., 1992. Kindling is associated with the formation of novel mossy ¢ber synapses in the CA3 region. Exp. Brain Res. 92, 69^78. Represa, A., Le Gall La Salle, G., Ben-Ari, Y., 1989. Hippocampal plasticity in the kindling model of epilepsy in rats. Neurosci. Lett. 99, 345^350.
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Represa, A., Niquet, J., Pollard, H., Khrestchatisky, M., Ben-Ari, Y., 1994. From seizures to neo-synaptogenesis : intrinsic and extrinsic determinants of mossy ¢ber sprouting in the adult hippocampus. Hippocampus 4, 270^274. Ribak, C.E., Tran, P.H., Spigelman, I., Okazaki, M.M., Nadler, J.V., 2000. Status epilepticus-induced hilar basal dendrites on rodent granule cells contribute to recurrent excitatory circuitry. J. Comp. Neurol. 428, 240^253. Santhakumar, V., Ratzli¡, A.D., Jeng, J., Toth, Z., Soltesz, I., 2001. Long-term hyperexcitability in the hippocampus after experimental head trauma. Ann. Neurol. 50, 708^717. Sato, M., Racine, R.J., McIntyre, D.C., 1990. Kindling : basic mechanisms and clinical validity. Electroenceph. Clin. Neurophysiol. 76, 459^472. Stan¢eld, B.B., 1989. Excessive intra- and supragranular mossy ¢ber in the dentate gyrus of tottering (tg/tg) mice. Brain Res. 480, 294^299. Stripling, J.S., Patneau, D.K., 1990. Seizure mechanisms in the piriform cortex. In: Wada, J.A. (Ed.), Kindling, Vol. 4. Plenum, New York, pp. 45^57. Sudhof, T.C., Lottspeich, F., Greengard, P., Mehl, E., Jahn, R., 1987. A synaptic vesicle protein with a novel cytoplasmic domain and four transmembrane regions. Science 238, 1142^1144. Sutula, T., Cascino, G., Cavazos, J., Parada, I., Ramirez, L., 1989. Mossy ¢ber synaptic reorganization in the epileptic human temporal lobe. Ann. Neurol. 26, 321^330. Sutula, T., He, X.X., Cavazos, J., Scott, G., 1988. Synaptic reorganization in the hippocampus induced by abnormal functional activity. Science 239, 1147^1150. Sutula, T.P., Golarai, G., Cavazos, J., 1992. Assessing the functional signi¢cance of mossy ¢ber sprouting. Epilepsy Res. 7, 251^259. Teskey, G.C., Racine, R.J., 1993. Increased spontaneous unit discharge rates following electrical kindling in the rat. Brain Res. 624, 11^18. Timofeeva, O.A., Peterson, G.M., 1997. Delayed development of spontaneous seizures and prolonged convulsive state in rats after massed stimulation of the anterior piriform cortex. Brain Res. 754, 227^238. Timofeeva, O.A., Peterson, G.M., 1999. Dissociation of mossy ¢ber sprouting and electrically-induced seizure sensitivity : rapid kindling versus adaptation. Epilepsy Res. 33, 99^115. Vaidya, V.A., Siuciak, J.A., Du, F., Duman, R.S., 1999. Hippocampal mossy ¢ber sprouting induced by chronic electroconvulsive seizures. Neuroscience 89, 157^166. Van der Zee, C.E.E.M., Rashid, K., Le, K., Moore, K., Stanisz, J., Diamond, J., Racine, R.J., Fahnestock, M., 1995. Intraventricular administration of antibodies to nerve growth factor retards kindling and blocks mossy ¢ber sprouting in adult rats. J. Neurosci. 15, 5316^5323. Watanabe, Y., Johnson, R.S., Butler, L.S., Binder, D.K., Spiegelman, B.M., Papaioannou, V.M., McNamara, J.O., 1996. Null mutation of c-fos impairs structural and functional plasticities in the kindling model of epilepsy. J. Neurosci. 16, 3827^3836. Wiedenmann, B., Franke, W.W., 1985. Identi¢cation and localization of synaptophysin, an integral membrane glycoprotein of Mr 38,000 characteristic of presynaptic vesicles. Cell 41, 1017^1028. (Accepted 17 July 2002)
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Neuroscience Vol. 115, No. 4, pp. 1221^1229, 2002 A 2002 IBRO. Published by Elsevier Science Ltd All rights reserved. Printed in Great Britain 0306-4522 / 02 $22.00+0.00
www.neuroscience-ibro.com
ACTIVITY-DEPENDENT CHANGES IN SYNAPTOPHYSIN IMMUNOREACTIVITY IN HIPPOCAMPUS, PIRIFORM CORTEX, AND ENTORHINAL CORTEX OF THE RAT S. LI,a;b I. REINPRECHT,c M. FAHNESTOCKb and R. J. RACINEa a b
Department of Psychology, McMaster University, 1280 Main Street West, Hamilton, ON, Canada L8S 4K1
Department of Psychiatry and Behavioral Neurosciences, McMaster University, Hamilton, ON, Canada L8N 3Z5 c
JSW-Research GmbH, Rankengasse 28, 8020 Graz, Austria
Abstract:Synaptophysin, an integral membrane glycoprotein of synaptic vesicles, has been widely used to investigate synaptogenesis in both animal models and human patients. Kindling is an experimental model of complex partial seizures with secondary generalization, and a useful model for studying activation-induced neural growth in adult systems. Many studies using Timm staining have shown that kindling promotes sprouting in the mossy ¢ber pathway of the dentate gyrus. In the present study, we used synaptophysin immunohistochemistry to demonstrate activation-induced neural sprouting in non-mossy ¢ber cortical pathways in the adult rat. We found a signi¢cant kindling-induced increase in synaptophysin immunoreactivity in the stratum radiatum of CA1 and stratum lucidum/radiatum of CA3, the hilus, the inner molecular layer of the dentate gyrus, and layer II/III of the piriform cortex, but no signi¢cant change in layer II/III of the entorhinal cortex, 4 weeks after the last kindling stimulation. We also found that synaptophysin immunoreactivity was lowest in CA3 near the hilus and increased with increasing distance from the hilus, a reverse pattern to that seen with Timm stains in stratum oriens following kindling. Furthermore, synaptophysin immunoreactivity was lowest in dorsal and greatest in ventral sections of both CA3 and dentate gyrus in both kindled and non-kindled animals. This demonstrates that di¡erent populations of sprouting axons are labeled by these two techniques, and suggests that activationinduced sprouting extends well beyond the hippocampal mossy ¢ber system. A 2002 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: axonal sprouting, immunohistochemistry, kindling.
widespread use of the Timm stain (Danscher, 1981; Danscher and Zimmer, 1978). The Timm stain has the major limitation that it labels only pathways containing heavy metals, most notably the mossy ¢ber pathway. Thus it is not clear whether there is activation-induced sprouting in other ¢ber tracts. Axonal sprouting can be measured more generally with biochemical markers of the nerve terminals such as synaptophysin (Masliah et al., 1990, 1993; Bergmann et al., 1997). Synaptophysin, which is a 38-kDa integral membrane glycoprotein, is a component of synaptic vesicles (Jahn et al., 1985; Wiedenmann and Franke, 1985; Sudhof et al., 1987). The precise function of synaptophysin is unclear, but it is involved in synaptic vesicle formation and exocytosis (Bergmann et al., 1993; Mundigl et al., 1993; Grabs et al., 1994; Papa et al., 1995; Papa and Segal, 1996). As a molecular marker for the presynaptic vesicle membrane, as well as a functional marker for synapses, synaptophysin has been widely used to investigate synaptogenesis in both animal models and human patients (Masliah et al., 1990, 1993; Alder et al., 1992; Mahata et al., 1992; Alford et al., 1994; Davies et al., 1998; Looney et al., 1999; Reinprecht et al., 1999; Hinz et al., 2001). Little is known about the relationship between the expression of synaptophysin immunoreactivity and electrical kindling, although synaptophysin immunohistochemistry
Sprouting of mossy ¢ber terminals is observed following seizures in human patients (Sutula et al., 1989; Ben-Ari and Represa, 1990; Houser, 1990) and in a variety of animal models including kainic acid- and pilocarpineinduced seizures (Longo and Mello, 1997; Ribak et al., 2000), head injury (Mathern et al., 1996; Golarai et al., 2001; Santhakumar et al., 2001), electroconvulsive shock (Gombos et al., 1999; Vaidya et al., 1999), long-term potentiation (Adams et al., 1997a), and kindling (Sutula et al., 1988; Represa et al., 1989, 1994; Stan¢eld, 1989; Sato et al., 1990; Represa and Ben-Ari, 1992; Rashid et al., 1995; Van der Zee et al., 1995; Golarai and Sutula, 1996; Watanabe et al., 1996; Adams et al., 1997b; Gombos et al., 1999). Kindling and long-term potentiation are used for studying activation-induced neural growth in adult systems, since these models can induce axonal sprouting in the absence of neural damage (Adams et al., 1997a, b). Axonal sprouting is most commonly studied in the mossy ¢ber pathway because of the
*Corresponding author. Tel. : +1-905-525-9140; fax: +1-905-5296225. E-mail address: [email protected] (R. J. Racine). Abbreviations : ANOVA, analysis of variance ; BSA, bovine serum albumin; IML, inner molecular layer ; PBS, phosphate-bu¡ered saline ; S.E.M., standard error of the mean. 1221
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has been used to demonstrate a connection between neural sprouting in the cortex and kainic acid-induced seizures (Chen et al., 1996) as well as in the hippocampus of epileptic patients (Davies et al., 1998; Proper et al., 2000). In this paper, we used synaptophysin immunohistochemistry to determine whether synaptogenesis can be induced in non-mossy ¢ber systems in the hippocampal formation and in the piriform and entorhinal cortices by electrical kindling.
EXPERIMENTAL PROCEDURES
Sections were then mounted on Superfrost-plus slides (Fisher Scienti¢c, USA) for immunohistochemistry as described previously (Reinprecht et al., 1999). In brief, mouse antiserum directed against rat synaptophysin (Boehringer Mannheim, ON, Canada) was diluted 1:50 in PBS containing 1.0% bovine serum albumin (BSA). Following blocking with 5% normal horse serum in PBS, slides were incubated with primary antibody overnight at 4‡C. Antigen/antibody complexes were visualized by the avidin^biotin^peroxidase complex technique (Hsu and Raine, 1981a; Hsu et al., 1981b) using biotinylated goat anti-mouse IgG (Vectastain, Vector Laboratories, CA, USA) diluted 1:100 in PBS^BSA and incubated for 30 min at room temperature, followed by 0.005% 3,3-diaminobenzidine-tetrahydrochloride (Vectastain, Vector Laboratories). Sections incubated without the primary antibody served as negative controls.
Animals
Densitometric analysis of synaptophysin immunoreactivity
Fourteen adult male Long^Evans hooded rats (Charles River, St. Constant, Quebec, Canada), weighing between 230 and 260 g at the time of surgery, were used in this study. All animal experiments were carried out in accordance with the Canadian Council on Animal Care Guide for the Care and Use of Laboratory Animals. E¡orts were made to minimize animal su¡ering and to minimize the number of animals used. Rats were housed individually, maintained on an ad libitum feeding schedule, and kept on a 12-h light/dark cycle.
To minimize the variability of synaptophysin-immunoreactive staining, sections from both groups of animals were stained simultaneously. Eight horizontal sections taken from each animal were examined at 50U magni¢cation using a Bioquant image analysis system (Bioquant-R and M Biometrics, Nashville, TN, USA) attached to a light microscope (Zeiss Axioskop, Oberkochen, Germany). To determine the density of the synaptophysinimmunoreactive label in the hippocampus, the optical densities of areas CA1 and CA3, dentate gyrus and background were measured by placing an open circular cursor with a diameter of 100 Wm at eight adjacent positions along the stratum radiatum of the area CA1 (Fig. 1A), 16 adjacent positions along the stratum lucidum/radiatum (starting at the hilus) of the area CA3 (Fig. 1B), and 10 adjacent positions along the hilus and inner molecular layer (IML) of the dentate gyrus, respectively (Fig. 1C). For the measurements taken from entorhinal and piriform cortices, three open circular cursors (diameter of 400 Wm) were placed along layer II/III of the entorhinal cortex (Fig. 1D) and three open rectangular cursors (1200 WmU300 Wm) were placed along layer II/III starting at the genu of the piriform cortex (Fig. 1E). For background staining, optical densities were measured within eight adjacent circular cursors of 100 Wm diameter placed along the corpus callosum. To control for variations in background synaptophysin-immunoreactive staining across sections, the average of eight background density readings from corpus callosum was subtracted from the density readings of each section. Analyses were done on both raw densitometry measurements and on measurements corrected for background density. Both the right and left sides of the brains were measured for all sections of each animal. In addition, to ensure that the optical density of synaptophysin immunohistochemical labeling accurately re£ects the quantity of synaptophysin-positive synapses, six adjacent circular cursors (diameter of 100 Wm) were placed across stratum lucidum/radiatum and molecular layers, perpendicular to CA3, and both the mean optical density of the synaptophysin-immunoreactive labeling and the number of synaptophysin-immunoreactive terminals were measured in the same six sections. The mean density values obtained were linearly correlated with the number of synaptophysin-immunoreactive sites (R2 = 0.9906, Fig. 2). The strength of the correlation suggests that the mean density measurements directly re£ect the quantity of synaptophysin-positive terminals.
Surgery and kindling procedures All rats were deeply anesthetized with sodium pentobarbital (65 mg/kg, i.p.) and treated with atropine sulfate (1 mg/kg, i.p.) to minimize tracheal secretions and stress responses. The animals were placed in a stereotaxic frame (David Kopf Instruments, Tujunga, CA, USA), and a bipolar electrode made from Te£on-coated, stainless steel wires (O.D. = 190 Wm) was implanted into the right perforant path (7.6 mm behind bregma; 4.1 mm lateral to the midline; 3.3 mm below the skull surface) (Paxinos and Watson, 1985). The electrode was held in place by dental cement and three stainless steel screws inserted into the skull. A fourth screw, with a male Amphenol pin attached, served as the ground electrode. Each animal was given 30 000 U of penicillin (i.m.) and oral Apo-sulfatrim following surgery. All animals were given 7 days for recovery from surgery before the initiation of the kindling protocol. Beginning on day 7 after surgery, the kindled rats received stimulations (twice daily for a total of 11 days) consisting of a 1-s train of 1-ms pulses at an intensity of 500^600 WA and a frequency of 60 Hz. This was su⁄cient to trigger an epileptiform afterdischarge after each stimulation, the duration and magnitude of which were determined from electroencephalographic recordings. Sham-stimulated animals were treated identically, but without any electrical stimulation. The behavioral progression of kindling-induced seizures was scored according to Racine’s standard classi¢cation (Racine, 1972): stage 1, mouth and facial twitches; stage 2, clonic head movements; stage 3, unilateral forelimb clonus followed by contralateral clonus; stage 4, clonic rearing; and stage 5, loss of postural control and falling. All seven kindled rats exhibited at least three consecutive stage 5 seizures by the end of the kindling procedure. Perfusion and synaptophysin immunohistochemistry Rats were deeply anesthetized with an overdose of urethane (1.25 mg/kg, i.p.) and perfused transcardially (descending aorta clamped) with 60 ml saline bu¡ered with 50 mM sodium phosphate (PBS), pH 7.4, followed by 200 ml 4% paraformaldehyde in PBS on day 46 postsurgery and/or on day 28 after the last kindling stimulation. The brains were removed, quickly frozen by immersing them in cold 2-methylbutane (Sigma) on dry ice and stored at 370‡C. After embedding the brains individually in OCT Tissue-Tek (Miles, Elkhart, IN, USA), horizontal, serial, 12-Wm sections were taken between 4.1 and 8.6 mm ventral to bregma (Paxinos and Watson, 1985) using a cryostat at 320‡C.
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Abbreviations used in the ¢gures DG ENT GC ML PC PIR SL SR
dentate gyrus entorhinal cortex granule cell layer molecular layer pyramidal cell layer piriform cortex stratum lucidum stratum radiatum
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Fig. 1. Digitized images of the hippocampal CA1 and CA3 ¢elds, dentate gyrus, and entorhinal and piriform cortices. Optical density measurements of synaptophysin immunoreactivity were performed by placing open circular cursors at eight adjacent postions along the stratum radiatum of CA1 (A), 16 adjacent positions along the stratum lucidum/radiatum of CA3 (starting from CA3c) (B), 10 adjacent positions along the IML and hilus of the dentate gyrus (C), three open circular cursors along layer II/III of the entorhinal cortex (D), and three open rectangular cursors along layer II/III starting at the genu of the piriform cortex (E), as described in the section on experimental procedures. DG, dentate gyrus; ENT, entorhinal cortex; PIR, piriform cortex. Scale bar = 100 Wm (A^D) and 300 Wm (E).
Data analysis
RESULTS
Four-way analysis of variance (ANOVA) (e.g. [4U(2U5U8)]) with one between variable (group) and three within variables [brain hemisphere (left or right), section (1^5 ventral to dorsal, in CA1, CA3, IML, and hilus; 1^3 ventral to dorsal, in piriform and entorhinal cortices), cursor position (1^8 in CA1; 1^16 in CA3; 1^10 in IML and hilus; 1^3 in piriform and entorhinal cortices)] was conducted for the analysis of synaptophysin immunoreactivity in the CA1, CA3, IML, hilus, piriform and entorhinal regions. The ANOVA was followed by post hoc Tukey comparisons. Data were considered signi¢cantly di¡erent at a minimum con¢dence level of P 6 0.05. All data reported in the present study are expressed as mean W standard error of the mean (S.E.M.).
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Synaptophysin immunoreactivity in CA1 and CA3 A four-way (groupU(brain hemisphereUlevelUcursor position)) ANOVA with one between and three within variables was conducted for the analysis of synaptophysin immunoreactivity in the stratum radiatum of CA1 and the stratum lucidum/radiatum of CA3. Analyses were done on measurements corrected for background density. Statistical analyses revealed no in£uence of seizure activity on the background staining in corpus callosum. The negative control slides were devoid of
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staining. The optical density of synaptophysin immunoreactivity in the CA1 ¢eld of the kindled animals was increased signi¢cantly, by approximately 48.9%, compared to the control animals [Fð1;12Þ = 18.25; P 6 0.005, Fig. 3A, B; Table 1]. In CA3, the density of synaptophysin immunoreactivity was also enhanced signi¢cantly, by approximately 51.5%, in the kindled groups relative to the non-kindled controls [Fð1;12Þ = 24.47; P 6 0.01, Figs. 3C, D and 4; Table 1]. Synaptophysin immunoreactivity was distributed in a gradient, with the optical density of synaptophysin immunoreactivity greatest in ventral and lowest in dorsal sections [Fð4;48Þ = 3.51; P 6 0.05, Fig. 5A]. The density of synaptophysin immunoreactivity in hilus and IML A three-way ANOVA (groupU(brain hemisphereU level)) was utilized to evaluate synaptophysin immunoreactivity in the hilus and IML of the dentate gyrus. There was a main e¡ect for group. Post hoc analysis revealed the synaptophysin immunoreactivity increased signi¢cantly, by 46.2%, in the hilus [Fð1;12Þ = 25.32, P 6 0.0005] and, by 56.9%, in the IML [Fð1;12Þ = 19.22, P 6 0.001] of kindled rats as compared with non-kindled rats (Fig. 3E, F; Table 1). In the IML, there was a main e¡ect for section. Post hoc Tukey tests showed that the optical density of synaptophysin immunoreactivity was greatest in ventral and lowest in dorsal sections of all animals [Fð4;48Þ = 3.32; P 6 0.05, Fig. 5B], which was similar to the distribution in the CA3 ¢eld. The density of synaptophysin immunoreactivity in piriform and entorhinal cortices A two-way (groupUbrain hemisphere) ANOVA was used to evaluate synaptophysin-immunoreactive presynaptic terminals in the piriform and entorhinal cortices. Post hoc Tukey tests showed an enhancement of 37.8% [Fð1;12Þ = 7.28, P 6 0.05] in the density of synaptophysin immunoreactivity in layer II/III in the piriform cortex of kindled rats as compared with controls (Fig. 3G, H; Table 1). However, there was no signi¢cant di¡erence in synaptophysin immunoreactivity in layer II/III in the entorhinal cortex between the kindled and non-kindled animals [Fð1;12Þ = 1.61, P s 0.05; Fig. 3I, J; Table 1].
Fig. 2. Plot of mean optical density of synaptophysin immunoreactivity (x-axis) vs. number of synaptophysin-immunoreactive sites (y-axis). These values were closely correlated in a linear manner.
DISCUSSION
The immunohistochemical labeling of synaptophysin provides a molecular marker for presynaptic vesicle membranes, allowing us to investigate the density of axon terminals in pathways that do not contain high levels of heavy metals, e.g. in the piriform and entorhinal cortices, as well as in previously studied hippocampal areas. In the present study, we demonstrate signi¢cant kindling-induced increases in synaptophysin immunoreactivity in the stratum radiatum of the CA1 and the stratum lucidum/radiatum of the CA3 ¢elds, the hilus, the IML of the dentate gyrus, and in layer II/III of the piriform cortex. No signi¢cant di¡erence was found in synaptophysin immunoreactivity in layer II/III of the entorhinal cortex of kindled rats compared to controls. We have demonstrated that the increased synaptophysinimmunoreactive optical density is directly correlated with the number of synaptophysin-positive terminals, suggesting that the kindling-induced increase in synaptophysin immunoreactivity re£ects sprouting of new terminals. However, we cannot rule out the possibility that kindling stimulation up-regulates synaptophysin protein in preexisting unlabeled terminals. Previous studies have reported no change in synaptophysin immunoreactivity following amygdala kindling (Hinz et al., 2001) or pentylenetetrazole kindling (Mahata et al., 1992). In those studies, synaptophysin immunohistochemistry was performed 2 days following the last stimulation. In the present study, we demonstrate that kindling increases synaptophysin immunoreactivity
Table 1. Mean density of synaptophysin immunoreactivity in hippocampus and cortices Group
n
Control
Kindled
CA1 CA3 Hilus IML Piriform (Wm32 ) Entorhinal (Wm32 )
7 7 7 7 7 7
18.62 W 0.81 18.53 W 0.79 23.94 W 1.27 19.58 W 1.60 0.037 W 0.00066 0.042 W 0.00063
27.73 W 1.60* 28.07 W 1.64* 35.01 W 2.74* 30.73 W 1.69* 0.051 W 0.00415* 0.043 W 0.00068
W S.E.M.; *P 6 0.05.
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Fig. 3. Representative synaptophysin-stained sections of CA1 (A, B), CA3 (C, D), dentate gyrus (E, F), piriform cortex (G, H), and entorhinal cortex (I, J). Sections from control rats are shown in A, C, E, G and I, and sections from kindled rats are shown in sections B, D, F, H and J. DG, dentate gyrus; ENT, entorhinal cortex; GC, granule cell layer; ML, molecular layer; PC, pyramidal cell layer ; PIR, piriform cortex; SL, stratum lucidum ; SR, stratum radiatum. Scale bar = 100 Wm.
in both the hippocampal formation and the piriform cortex 28 days following the last kindling stimulation. Mossy ¢ber sprouting is not detectable until at least 4 days following stimulation and is detectable over a period of months (Cavazos et al., 1991). Therefore, it is likely that previous studies failed to detect changes in synaptophysin-immunoreactive protein because sprouting is not yet evident 2 days after activation.
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Patterns of synaptophysin immunoreactivity compared to Timm stain The terminals of the mossy ¢ber axons that originate from granule cells of the dentate gyrus contain high levels of zinc, making the Timm stain an excellent means to track changes in the mossy ¢ber system. However, this technique is not e¡ective for monitoring axonal sprout-
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Fig. 3 (G^J).
ing outside the mossy ¢ber system. Our previous studies showed that Timm granule density increased markedly in the stratum oriens of CA3 and in the IML of the dentate gyrus of kindled rats compared to controls (Rashid et al., 1995; Van der Zee et al., 1995; Adams et al., 1997a,b). In the present study, we demonstrate that the optical density of synaptophysin immunoreactivity is also increased signi¢cantly in the stratum radiatum of CA1 and the stratum lucidum/radiatum of CA3, as well as in the hilus and IML of the dentate gyrus in kindled animals. Synaptic reorganization of the mossy ¢ber pathway following seizures in kindled animals is often associated with the development, progression, and permanence of kindling-induced seizures (Sutula et al., 1988, 1992; Cavazos et al., 1991; Dudek et al., 1994; Represa et al., 1994). Our experimental results indicate that kindling-induced seizure activity is also correlated with sprouting in other hippocampal pathways as measured by an independent molecular marker. While Timm granule density is greatest in the stratum oriens of CA3 near the hilus and decreases with increasing distance from the hilus (Rashid et al., 1995; Van der Zee et al., 1995; Adams et al., 1997b), synaptophysin immunoreactivity is lowest in the stratum lucidum/radiatum of CA3 near the hilus and increases with increasing distance from the hilus in kindled animals. Also, Timm granule density in CA3 is reliably greater in dorsal com-
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pared to ventral sections (Van der Zee et al., 1995; Adams et al., 1997b), whereas synaptophysin immunoreactivity is greatest in ventral and lowest in dorsal sections. The graded distribution of synaptophysin immunoreactivity in IML of the dentate gyrus is similar to that in the stratum lucidum/radiatum of area CA3. The di¡erent characteristics of synaptophysin immunoreactivity and Timm labeling in CA3 likely re£ects the labeling of di¡erent ¢ber and cell types within di¡erent layers or locations of hippocampus.
Fig. 4. Mean synaptophysin-immunoreactive optical density in the stratum lucidum/radiatum of CA3. Synaptophysin immunoreactivity was enhanced signi¢cantly in the kindled group (n = 7) relative to the non-kindled control group (P 6 0.01, n = 7). Error bars represent S.E.M.
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interictal spikes in the kindling model (Racine et al., 1988; McIntyre and Plant, 1989; McIntyre and Kelly, 1990; Stripling and Patneau, 1990; Haberly and Sutula, 1992; Teskey and Racine, 1993), but also one of the fastest brain sites for the progression of electrical kindling (Goddard et al., 1969; Racine, 1975; Cain, 1977). Recent studies with kindling and convulsant drug models of epilepsy suggest that the piriform cortex may be particularly susceptible to generation of epileptiform activity (Loscher and Ebert, 1996; Timofeeva and Peterson, 1997, 1999; Potschka et al., 2000). Kindling causes a marked and spontaneous ¢ring in neurons located at the transition between piriform cortex layers II and III (Gernert et al., 2000). Our ¢nding that electrical kindling up-regulates synaptophysin immunoreactivity in piriform cortex, but not in entorhinal cortex, is consistent with these results. It remains to be determined whether synaptic reorganization plays a critical role in the piriform cortex’s contribution to epileptogenesis. Conclusion
Fig. 5. Graded distribution of synaptophysin immunoreactivity according to section in kindled and non-kindled animals. (A) CA3, (B) IML. Synaptophysin immunoreactivity was greatest in ventral and lowest in dorsal sections (P 6 0.05, n = 14). Error bars represent S.E.M.
Synaptic sprouting in the piriform cortex and the role of the piriform cortex in kindling We ¢nd that the average terminal density as measured by synaptophysin immunoreactivity in layer II/III of piriform cortex is increased signi¢cantly in kindled rats, whereas no signi¢cant di¡erence is found in the entorhinal cortex between kindled and control rats. The piriform cortex is reportedly not only a primary locus for the generation of spontaneous, epileptiform,
We have found that kindling up-regulates synaptophysin immunoreactivity in the stratum radiatum of area CA1, the stratum lucidum/radiatum of area CA3, hilus, IML of the dentate gyrus, and piriform cortex 28 days following stimulation, but has no signi¢cant e¡ects in entorhinal cortex of the adult rat. It appears that seizure-induced synaptic reorganization extends well beyond the hippocampal mossy ¢ber sprouting shown with Timm stain. The piriform cortex, in particular, is an active site according to synaptophysin-immunoreactive staining. Even in the termination ¢elds of the mossy ¢bers, synaptophysin immunoreactivity revealed di¡erent gradients than were found with Timm labeling. This suggests that di¡erent populations of sprouting axons are labeled by the two techniques. Acknowledgements+This work was supported by Grant # MOP-13248 from the Canadian Institutes of Health Research to M.F. and R.J.R. I.R. was supported by a scholarship of the O⁄ce of International Relations of the Karl-Franzens University, Graz, Austria.
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