- Email: [email protected]
Time course for kindling-induced changes in the hilar area of the dentate gyrus: reactive gliosis as a potential mechanism
Brain Research 804 Ž1998. 331–336
Short communication
Time course for kindling-induced changes in the hilar area of the dentate gyrus: reactive gliosis as a potential mechanism Beth Adams a , Ee Von Ling a , Liezanne Vaccarella a , Gwen O. Ivy b , Margaret Fahnestock c , Ronald J. Racine a, ) a
Department of Psychology, McMaster UniÕersity, Hamilton, Ontario, Canada L8S 4K1 DiÕision of Life Sciences, UniÕersity of Toronto, Scarborough, Ontario, Canada M1C 1A4 Department of Biomedical Sciences, McMaster UniÕersity, Hamilton, Ontario, Canada L8S 4K1 b
c
Accepted 2 June 1998
Abstract Recurrent seizure activity induced during kindling has been reported to cause an increase in the hilar area of the dentate gyrus of the hippocampus. To date, very little is known about the mechanism of this increase. This study investigated the time course for kindling-induced changes in the hilar area of the dentate gyrus at seven days, one month, and two months post-kindling. Hilar area of the dentate gyrus was significantly increased by approximately 46% at seven days and remained elevated at one month, but declined back to control levels by two months. Glial fibrillary acidic protein ŽGFAP. immunostaining was also evaluated at the same time points to determine whether kindling-induced changes in the hilar area of the dentate gyrus are related to kindling-induced glial cell changes. Increases in hilar GFAP immunostaining by approximately 57% were observed at seven days and at one month post-kindling, but not at two months post-kindling. These findings indicate that kindling-induced changes in the hilar area of the dentate gyrus and kindling-induced glial cell changes follow a similar time course, and that kindling-induced glial cell changes may mediate the observed changes in the hilar area of the dentate gyrus. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Kindling; Gliosis; Hippocampus; Hilus; Seizure; Plasticity; Dentate gyrus
Kindling can be defined as the progressive increase in electrographic and behavioral seizure activity produced by spaced and repeated electrical stimulation of certain forebrain structures w6x. It has been well-documented that kindling produces a variety of changes in the hippocampal region of the brain, including sprouting of the mossy fiber pathway Ži.e., axons of the dentate granule cells. w4,12x and a decrease in neuronal density, particularly in the hilus of the dentate gyrus w3,5x. Kindling also causes another type of structural change in the hippocampus: an increase in the size of the hilar region of the dentate gyrus w1,2,14x. To date, however, potential mechanisms underlying this effect have not been investigated. From this point onwards, the hilar region of the dentate gyrus will be referred to as either the hilus or the hilar area. One possibility is that kindling-induced changes in the hilar area may be related to reactive gliosis w2x. Reactive
) Corresponding author. Fax: q1-905-529-6225; E-mail: [email protected]
gliosis is generally characterized by: Ž1. the proliferation and hypertrophy of glial cell bodies and processes and Ž2. the dramatic increases in the levels of glial fibrillary acidic protein ŽGFAP. and GFAP mRNA w13x. Furthermore, increased GFAP immunostaining is considered to be the biochemical hallmark denoting the transformation of normal glial cells to reactive glial cells w13x. Recent research has demonstrated that kindling up-regulates GFAP mRNA and protein levels in a time-dependent manner w7,13x, and that kindling causes glial cell hypertrophy and proliferation w8x. It has also been reported that kindling-induced reactive gliosis can occur in the absence of neuronal loss or degeneration w11x. The objectives in this study were two-fold: Ž1. to determine the time course for kindling-induced changes in the hilar area in adult rats and Ž2. to evaluate whether kindling-induced reactive gliosis could account for kindling-induced changes in the hilar area. Hilar area and reactive gliosis were evaluated at seven days, one month, and two months post-kindling compared to non-kindled, implanted controls. Specifically, we were interested in
0006-8993r98r$ - see front matter q 1998 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 8 . 0 0 6 0 5 - 2
332
B. Adams et al.r Brain Research 804 (1998) 331–336
determining whether kindling-induced reactive gliosis and kindling-induced hilar area changes follow a similar time course. Adult male Long–Evans hooded rats Ž n s 20. weighing between 300–350 g were used in these experiments. Rats were maintained on an ad lib feeding schedule, housed individually, and kept on a 12 h onr12 h off light cycle. Using stereotaxic procedures, rats were anaesthetized with sodium pentobarbitol Ž65 mgrkg. and were implanted with a bipolar electrode made from teflon-coated stainless steel wires in the right perforant path. Stereotaxic coordinates for the perforant path were 7.6 mm posterior and 4.1 mm lateral to bregma, and 3.3 mm ventral to the brain surface. The electrode was held in place by dental acrylic and three stainless steel screws inserted into the skull. Following a two-week recovery period, rats were randomly assigned to either a kindled Ž n s 15. or a non-kindled Ž n s 5. group. Non-kindled control rats remained in the colony for 32 days post-surgery. Rats in the kindled group received a 1-s train of 1-ms pulses at a frequency of 60 Hz and pulse intensity of 500–700 mA twice a day for 11 days. Progression of kindling was monitored behaviourally using Racine’s
seizure classification scale w10x and electrophysiologically using an electroencephalogram of the evoked epileptiform afterdischarges ŽAD.. Each stimulation evoked an AD of greater than 5 s. Each rat received a total of 22 afterdischarges. Following kindling, rats were randomly assigned to one of three groups: seven-day Ž n s 5., one-month Ž n s 5. and two-month Ž n s 5. groups. Kindled rats remained in the colony for the assigned time period following the last kindling stimulation. As expected, repeated-measures ANOVA confirmed that behavioral seizure stage increased as a function of stimulation number across all groups Ž p - 0.05; data not shown., and there was no significant difference in the behavioral progression of kindling between the groups Ž p ) 0.05.. Also, AD duration increased as a function of stimulation number across all three kindled groups Ž p - 0.05; data not shown., but did not differ among the three groups. These data suggest that there were no differences in either the behavioral or the electrographic progression of kindling between the three kindled groups. Therefore, any subsequent differences between the groups cannot be attributed to differences in response to the kindling procedure.
Fig. 1. Schematic of the measurement of the hilar area of the dentate gyrus. Hilar area was outlined by a thick line using the MCID image analysis system. Hilar area was defined by the inner edge of the granule cell layer and the lines connecting the tips of the two granule cell blades to the beginning of the pyramidal cell layer of Ammon’s horn.
B. Adams et al.r Brain Research 804 (1998) 331–336
333
temperature. After washing in PB, sections were then incubated with Vectastain ABC reagent ŽVector Laboratories. for 45 min at room temperature. Finally, after wash-
Fig. 2. Mean hilar area of the dentate gyrus as a function of treatment condition. A three-way ANOVA with subsequent post-hoc comparisons showed that mean hilar area was significantly increased at one week post-kindling and remained elevated at one month post-kindling Ž p0.05., but declined back to control levels at two months post-kindling Ž p- 0.05.. Mean hilar area was not significantly different between either the one-week and the one-month groups Ž p) 0.05., or between the control and the two-month groups Ž p) 0.05.. Values represent mean hilar area expressed in mm2 "S.E.M. ) P - 0.05.
Following the assigned time period, rats were perfused with 200 ml of 0.1 M phosphate buffered 0.85% saline ŽPBS; pH s 7.4. followed by 4% paraformaldehyde in 0.1 M phosphate buffer ŽPB. at 48C. Brains were post-fixed in 4% paraformaldehyde in PB for 24 h and then stored in a 20% sucrose solution Ž20 g sucroser100 g 0.1 M PB. for 24 h at 48C for cryoprotection. Brains were subsequently frozen in isopentane cooled to y408C. Horizontal serial 30-mm sections were cut using a sliding microtome and section depth was determined using an atlas, The Rat Brain in Stereotaxic Coordinates w9x. To ensure that brain sections included in the data analysis were from comparable levels, we selected six pairs of adjacent sections Ž12 sectionsrbrain. of the hippocampal area at 4.1–7.1 mm ventral to bregma and 600 mm apart to be processed for immunocytochemistry. Sections were incubated overnight with monoclonal anti-GFAP Žclone G-A-5; Boehringer Mannheim, Laval, Quebec, Canada; 1:200. at 48C. After washing in 0.1 M phosphate buffer, sections were incubated with biotinylated anti-mouse IgG ŽBA-2000; Dimension Laboratories, Mississauga, Ontario, Canada; 1:200. for 1 h at room
Fig. 3. Photomicrographs Ž445 mm=285 mm. of GFAP immunostaining in the hilar region at 200= magnification. Representative examples of GFAP immunostained sections in a control rat ŽA., in a rat at seven days post-kindling ŽB., in a rat at one month post-kindling ŽC. and in a rat at two months post-kindling ŽD.. Note that reactive gliosis is evident in both the one-week and one-month groups, but is not apparent in either the control or the two-month groups.
334
B. Adams et al.r Brain Research 804 (1998) 331–336
Fig. 4. MCID images of GFAP immunostained hilar fields Ž200 mm = 160 mm. before ŽA. and after ŽB. density thresholding, as indicated by the blue colour overlay. Note that after density thresholding, only blue target regions contribute to the proportional area measurement of GFAP immunostaining for each hilar field.
B. Adams et al.r Brain Research 804 (1998) 331–336
ing in PB, sections were pre-incubated for 5 min with 70 mg of diaminobenzidine ŽSigma. in 100 ml of 0.1 M PB and then incubated in this solution for 60–90 s by adding 30 ml of 30% H 2 O 2 , until the desired staining intensity developed. To ensure comparable levels of immunostaining, tissue from all groups was always batchprocessed. Following immunostaining, tissue sections were mounted on chrom alum-coated slides. One section from each adjacent pair of sections Žsix sectionsrbrain. was also counterstained with Cresyl violet for the determination of the hilar area. Slides were then coded and all subsequent analyses were conducted by an observer who was unaware of the treatment of the animal to ensure objectivity in the data analysis. For the evaluation of the hilar area, horizontal sections immunostained with GFAP and counterstained with Cresyl violet were examined at 50 = magnification by creating a digitized image with a Micro Computer Imaging Device ŽMCID. ŽBrock University, St. Catherine’s, Ontario, Canada. attached to a light microscope ŽZeiss Axioskop, Oberkochen, Germany. with a high-resolution charge-couple device ŽCCD. camera ŽMTI CCD 72.. Hilar area was defined by the inner edge of the granule cell layer and the lines connecting the tips of the two granule cell blades to the beginning of the pyramidal cell layer of Ammon’s horn w2x ŽFig. 1.. A Ž4 = Ž2 = 6.. ANOVA Žgroup = Žbrain hemisphere= level.. was conducted to evaluate the hilar area. There was a main effect for group: F Ž3,16. s 22.22; p - 0.001 ŽFig. 2.. Post-hoc Tukey tests showed that mean hilar area was significantly increased by approximately 47% at one week post-kindling Ž p - 0.01. and remained elevated at one month post-kindling Ž p - 0.01., but declined back to control levels by two months post-kindling Ž p - 0.01.. There were no significant differences between the one-week and one-month kindled groups Ž p ) 0.05. or between the control group and the two-month kindled group Ž p - 0.05.. These findings suggest that kindling-induced increases in the hilar area are not permanent. Fig. 3 shows representative examples of GFAP immunostained sections in a control rat ŽA., in rats at seven days ŽB., one month post-kindling ŽC. and two months post-kindling ŽD.. These sections clearly show increases in glial cell size in the seven-day and one-month groups. Given that increased GFAP immunostaining is considered to reflect reactive gliosis w13x, hilar GFAP immunostaining was used to quantify reactive gliosis in the present study. Horizontal sections immunostained with GFAP were examined at 400 = magnification using MCID. Hilar GFAP immunostaining was evaluated in a hilar field Ž0.2 mm = 0.48 mm. starting at the hilar end of the CA3rCA4 for each brain section, using MCID’s target detection feature. This feature permits image components to be separated into valid targets and background based on the optical density of the target. Glial cell bodies and processes were regarded as valid targets in the present study. A target
335
acceptance criteria was established using a segmentation range between the upper and lower density thresholds of the target. That is, pixels lying within the segmentation range were regarded as valid targets, whereas pixels lying outside of the range were ignored as background. The segmentation range was set by manually decreasing the thresholding value until a blue overlay display, designating the thresholded area, completely occupied the glial cell bodies and processes Ži.e., the target., but not the background. Fig. 4 shows MCID images of GFAP immunostained hilar fields before ŽA. and after ŽB. density thresholding. The mean proportional area Ži.e., the proportion of the field that is occupied by the target. of GFAP immunostaining was calculated for each hilar field per section. A Ž4 = Ž2 = 6.. ANOVA Žgroup = Žbrain hemisphere= level.. was conducted to evaluate the mean proportional area of hilar GFAP immunostaining. There was a main effect for group: F Ž3,16. s 44.24; p - 0.0001 ŽFig. 5.. Post-hoc Tukey tests showed that the mean proportional area of GFAP immunostaining was significantly increased by approximately 57% at seven days post-kindling, remained elevated at one month post-kindling, but declined back to control levels at two months post-kindling. There were no differences between the seven-day and one-month post-kindling groups Ž p ) 0.05., and there were no differences between the control and the two-month post-kindling groups Ž p ) 0.05.. These findings are similar to those obtained by Hansen et al. Ž1991., who demonstrated that GFAP protein levels remained elevated in most limbic
Fig. 5. Mean proportional area of hilar GFAP immunostaining as a function of treatment condition. A three-way ANOVA with subsequent post-hoc comparisons showed that mean area GFAP immunostaining as a proportion of the total field was significantly elevated at one week post-kindling and remained elevated at one month post-kindling Ž p0.05., but decreased to control levels by two months post-kindling Ž p- 0.05.. Mean proportional area of GFAP immunostaining was not significantly different between either the one-week and the one-month groups Ž p) 0.05., or between the control and the two-month groups Ž p) 0.05.. ) P - 0.05.
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structures at one week post-kindling, but declined to control levels at two months post-kindling w7x. These data suggest that kindling-induced up-regulation of GFAP is transient. The findings in the present study provide the first evidence that kindling-induced changes in the hilar area of the dentate gyrus and kindling-induced changes in GFAP immunostaining levels follow a similar time course. In addition, these findings provide support for the hypothesis that kindling-induced hilar area changes are mediated by kindling-induced glial cell changes. To date, however, the signal which leads to increased GFAP expression following kindling is unclear, and the role of reactive gliosis in kindling remains to be elucidated w11x. Additional research is required not only to define the relevance of glial cell mechanisms in the kindling process, but also to elucidate the role that kindling-induced reactive gliosis plays in mediating kindling-induced hilar area changes in the dentate gyrus.
Acknowledgements
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This work was supported by grants from the Neuroscience Network Centers of Excellence ŽNCE. ŽR.J.R. and M.F.. and the Medical Research Council of Canada ŽMRC. ŽR.J.R. and M.F... B.A. was supported by a studentship from the Savoy Foundation and a supplement from the NCE.
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References w1x B. Adams, M. Sazgar, P. Osehobo, C.E.E.M. Van der Zee, J. Diamond, M. Fahnestock, R.J. Racine, Nerve growth factor acceler-
w14x
ates seizure development, enhances mossy fiber sprouting and attenuates seizure-induced decreases in neuronal density in the kindling model of epilepsy, J. Neurosci. 17 Ž14. Ž1997. 5288–5296. E.H. Bertram, E.W. Lothman, Morphometric effects of intermittent kindled seizures and limbic status epilepticus in the dentate gyrus of the rat, Brain Res. 603 Ž1993. 25–31. J.E. Cavazos, T.P. Sutula, Progressive neuronal loss induced by kindling: a possible mechanism for mossy fiber synaptic reorganization and hippocampal sclerosis, Brain Res. 527 Ž1990. 1–6. J.E. Cavazos, G. Golarai, T. Sutula, Mossy fiber sprouting reorganization induced by kindling: time course, development, progression and permanence, J. Neurosci. 11 Ž1991. 2795–2803. J.E. Cavazos, I. Das, T.P. Sutula, Neuronal loss induced in limbic pathways by kindling: evidence for induction of hippocampal sclerosis by repeated brief seizures, J. Neurosci. 14 Ž1994. 3106–3121. G. Goddard, D. McIntyre, C. Leech, A permanent change in brain function resulting from daily electrical stimulation, Exp. Neurol. 245 Ž1969. 745–761. A. Hansen, O. Steen Jorgensen, T.G. Bolwig, D.I. Barry, Hippocampal kindling in the rat is associated with time-dependent increases in the concentration of glial fibrillary acidic protein, J. Neurochem. 57 Ž5. Ž1991. 1716–1720. M. Khurgel, R.J. Racine, G.O. Ivy, Kindling causes changes in the composition of the astrocytic skeleton, Brain Res. 592 Ž1992. 338– 342. G. Paxinos, C. Watson, The Rat Brain in Stereotaxic Coordinates, 2nd edn., Academic Press, Sydney. R.J. Racine, Modification of seizure activity by electrical stimulation: II. Motor seizure, Electroencephalogr. Clin. Neurophysiol. 32 Ž1972. 281–294. O. Steward, E.R. Torre, R. Tomasulo, E. Lothman, Neuronal activity up-regulates astroglial gene expression, PNAS 88 Ž1991. 6819–6823. T. Sutula, X.X. He, J. Cavazos, G. Scott, Synaptic reorganization in the hippocampus induced by abnormal functional activity, Science 239 Ž1988. 1147–1150. E.R. Torre, E. Lothman, O. Steward, Glial response to neuronal activity: GFAP mRNA and protein levels are transiently increased in the hippocampus after seizures, Brain Res. 631 Ž1993. 256–264. Y. Watanabe, R.S. Johnson, L.S. Butler, D.K. Binder, B.M. Spiegelman, V.M. Papaioannou, J.O. McNamara, Null mutation of c-fos impairs structural and functional plasticities in the kindling model of epilepsy, J. Neurosci. 16 Ž1996. 3827–3836.
Short communication
Time course for kindling-induced changes in the hilar area of the dentate gyrus: reactive gliosis as a potential mechanism Beth Adams a , Ee Von Ling a , Liezanne Vaccarella a , Gwen O. Ivy b , Margaret Fahnestock c , Ronald J. Racine a, ) a
Department of Psychology, McMaster UniÕersity, Hamilton, Ontario, Canada L8S 4K1 DiÕision of Life Sciences, UniÕersity of Toronto, Scarborough, Ontario, Canada M1C 1A4 Department of Biomedical Sciences, McMaster UniÕersity, Hamilton, Ontario, Canada L8S 4K1 b
c
Accepted 2 June 1998
Abstract Recurrent seizure activity induced during kindling has been reported to cause an increase in the hilar area of the dentate gyrus of the hippocampus. To date, very little is known about the mechanism of this increase. This study investigated the time course for kindling-induced changes in the hilar area of the dentate gyrus at seven days, one month, and two months post-kindling. Hilar area of the dentate gyrus was significantly increased by approximately 46% at seven days and remained elevated at one month, but declined back to control levels by two months. Glial fibrillary acidic protein ŽGFAP. immunostaining was also evaluated at the same time points to determine whether kindling-induced changes in the hilar area of the dentate gyrus are related to kindling-induced glial cell changes. Increases in hilar GFAP immunostaining by approximately 57% were observed at seven days and at one month post-kindling, but not at two months post-kindling. These findings indicate that kindling-induced changes in the hilar area of the dentate gyrus and kindling-induced glial cell changes follow a similar time course, and that kindling-induced glial cell changes may mediate the observed changes in the hilar area of the dentate gyrus. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Kindling; Gliosis; Hippocampus; Hilus; Seizure; Plasticity; Dentate gyrus
Kindling can be defined as the progressive increase in electrographic and behavioral seizure activity produced by spaced and repeated electrical stimulation of certain forebrain structures w6x. It has been well-documented that kindling produces a variety of changes in the hippocampal region of the brain, including sprouting of the mossy fiber pathway Ži.e., axons of the dentate granule cells. w4,12x and a decrease in neuronal density, particularly in the hilus of the dentate gyrus w3,5x. Kindling also causes another type of structural change in the hippocampus: an increase in the size of the hilar region of the dentate gyrus w1,2,14x. To date, however, potential mechanisms underlying this effect have not been investigated. From this point onwards, the hilar region of the dentate gyrus will be referred to as either the hilus or the hilar area. One possibility is that kindling-induced changes in the hilar area may be related to reactive gliosis w2x. Reactive
) Corresponding author. Fax: q1-905-529-6225; E-mail: [email protected]
gliosis is generally characterized by: Ž1. the proliferation and hypertrophy of glial cell bodies and processes and Ž2. the dramatic increases in the levels of glial fibrillary acidic protein ŽGFAP. and GFAP mRNA w13x. Furthermore, increased GFAP immunostaining is considered to be the biochemical hallmark denoting the transformation of normal glial cells to reactive glial cells w13x. Recent research has demonstrated that kindling up-regulates GFAP mRNA and protein levels in a time-dependent manner w7,13x, and that kindling causes glial cell hypertrophy and proliferation w8x. It has also been reported that kindling-induced reactive gliosis can occur in the absence of neuronal loss or degeneration w11x. The objectives in this study were two-fold: Ž1. to determine the time course for kindling-induced changes in the hilar area in adult rats and Ž2. to evaluate whether kindling-induced reactive gliosis could account for kindling-induced changes in the hilar area. Hilar area and reactive gliosis were evaluated at seven days, one month, and two months post-kindling compared to non-kindled, implanted controls. Specifically, we were interested in
0006-8993r98r$ - see front matter q 1998 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 8 . 0 0 6 0 5 - 2
332
B. Adams et al.r Brain Research 804 (1998) 331–336
determining whether kindling-induced reactive gliosis and kindling-induced hilar area changes follow a similar time course. Adult male Long–Evans hooded rats Ž n s 20. weighing between 300–350 g were used in these experiments. Rats were maintained on an ad lib feeding schedule, housed individually, and kept on a 12 h onr12 h off light cycle. Using stereotaxic procedures, rats were anaesthetized with sodium pentobarbitol Ž65 mgrkg. and were implanted with a bipolar electrode made from teflon-coated stainless steel wires in the right perforant path. Stereotaxic coordinates for the perforant path were 7.6 mm posterior and 4.1 mm lateral to bregma, and 3.3 mm ventral to the brain surface. The electrode was held in place by dental acrylic and three stainless steel screws inserted into the skull. Following a two-week recovery period, rats were randomly assigned to either a kindled Ž n s 15. or a non-kindled Ž n s 5. group. Non-kindled control rats remained in the colony for 32 days post-surgery. Rats in the kindled group received a 1-s train of 1-ms pulses at a frequency of 60 Hz and pulse intensity of 500–700 mA twice a day for 11 days. Progression of kindling was monitored behaviourally using Racine’s
seizure classification scale w10x and electrophysiologically using an electroencephalogram of the evoked epileptiform afterdischarges ŽAD.. Each stimulation evoked an AD of greater than 5 s. Each rat received a total of 22 afterdischarges. Following kindling, rats were randomly assigned to one of three groups: seven-day Ž n s 5., one-month Ž n s 5. and two-month Ž n s 5. groups. Kindled rats remained in the colony for the assigned time period following the last kindling stimulation. As expected, repeated-measures ANOVA confirmed that behavioral seizure stage increased as a function of stimulation number across all groups Ž p - 0.05; data not shown., and there was no significant difference in the behavioral progression of kindling between the groups Ž p ) 0.05.. Also, AD duration increased as a function of stimulation number across all three kindled groups Ž p - 0.05; data not shown., but did not differ among the three groups. These data suggest that there were no differences in either the behavioral or the electrographic progression of kindling between the three kindled groups. Therefore, any subsequent differences between the groups cannot be attributed to differences in response to the kindling procedure.
Fig. 1. Schematic of the measurement of the hilar area of the dentate gyrus. Hilar area was outlined by a thick line using the MCID image analysis system. Hilar area was defined by the inner edge of the granule cell layer and the lines connecting the tips of the two granule cell blades to the beginning of the pyramidal cell layer of Ammon’s horn.
B. Adams et al.r Brain Research 804 (1998) 331–336
333
temperature. After washing in PB, sections were then incubated with Vectastain ABC reagent ŽVector Laboratories. for 45 min at room temperature. Finally, after wash-
Fig. 2. Mean hilar area of the dentate gyrus as a function of treatment condition. A three-way ANOVA with subsequent post-hoc comparisons showed that mean hilar area was significantly increased at one week post-kindling and remained elevated at one month post-kindling Ž p0.05., but declined back to control levels at two months post-kindling Ž p- 0.05.. Mean hilar area was not significantly different between either the one-week and the one-month groups Ž p) 0.05., or between the control and the two-month groups Ž p) 0.05.. Values represent mean hilar area expressed in mm2 "S.E.M. ) P - 0.05.
Following the assigned time period, rats were perfused with 200 ml of 0.1 M phosphate buffered 0.85% saline ŽPBS; pH s 7.4. followed by 4% paraformaldehyde in 0.1 M phosphate buffer ŽPB. at 48C. Brains were post-fixed in 4% paraformaldehyde in PB for 24 h and then stored in a 20% sucrose solution Ž20 g sucroser100 g 0.1 M PB. for 24 h at 48C for cryoprotection. Brains were subsequently frozen in isopentane cooled to y408C. Horizontal serial 30-mm sections were cut using a sliding microtome and section depth was determined using an atlas, The Rat Brain in Stereotaxic Coordinates w9x. To ensure that brain sections included in the data analysis were from comparable levels, we selected six pairs of adjacent sections Ž12 sectionsrbrain. of the hippocampal area at 4.1–7.1 mm ventral to bregma and 600 mm apart to be processed for immunocytochemistry. Sections were incubated overnight with monoclonal anti-GFAP Žclone G-A-5; Boehringer Mannheim, Laval, Quebec, Canada; 1:200. at 48C. After washing in 0.1 M phosphate buffer, sections were incubated with biotinylated anti-mouse IgG ŽBA-2000; Dimension Laboratories, Mississauga, Ontario, Canada; 1:200. for 1 h at room
Fig. 3. Photomicrographs Ž445 mm=285 mm. of GFAP immunostaining in the hilar region at 200= magnification. Representative examples of GFAP immunostained sections in a control rat ŽA., in a rat at seven days post-kindling ŽB., in a rat at one month post-kindling ŽC. and in a rat at two months post-kindling ŽD.. Note that reactive gliosis is evident in both the one-week and one-month groups, but is not apparent in either the control or the two-month groups.
334
B. Adams et al.r Brain Research 804 (1998) 331–336
Fig. 4. MCID images of GFAP immunostained hilar fields Ž200 mm = 160 mm. before ŽA. and after ŽB. density thresholding, as indicated by the blue colour overlay. Note that after density thresholding, only blue target regions contribute to the proportional area measurement of GFAP immunostaining for each hilar field.
B. Adams et al.r Brain Research 804 (1998) 331–336
ing in PB, sections were pre-incubated for 5 min with 70 mg of diaminobenzidine ŽSigma. in 100 ml of 0.1 M PB and then incubated in this solution for 60–90 s by adding 30 ml of 30% H 2 O 2 , until the desired staining intensity developed. To ensure comparable levels of immunostaining, tissue from all groups was always batchprocessed. Following immunostaining, tissue sections were mounted on chrom alum-coated slides. One section from each adjacent pair of sections Žsix sectionsrbrain. was also counterstained with Cresyl violet for the determination of the hilar area. Slides were then coded and all subsequent analyses were conducted by an observer who was unaware of the treatment of the animal to ensure objectivity in the data analysis. For the evaluation of the hilar area, horizontal sections immunostained with GFAP and counterstained with Cresyl violet were examined at 50 = magnification by creating a digitized image with a Micro Computer Imaging Device ŽMCID. ŽBrock University, St. Catherine’s, Ontario, Canada. attached to a light microscope ŽZeiss Axioskop, Oberkochen, Germany. with a high-resolution charge-couple device ŽCCD. camera ŽMTI CCD 72.. Hilar area was defined by the inner edge of the granule cell layer and the lines connecting the tips of the two granule cell blades to the beginning of the pyramidal cell layer of Ammon’s horn w2x ŽFig. 1.. A Ž4 = Ž2 = 6.. ANOVA Žgroup = Žbrain hemisphere= level.. was conducted to evaluate the hilar area. There was a main effect for group: F Ž3,16. s 22.22; p - 0.001 ŽFig. 2.. Post-hoc Tukey tests showed that mean hilar area was significantly increased by approximately 47% at one week post-kindling Ž p - 0.01. and remained elevated at one month post-kindling Ž p - 0.01., but declined back to control levels by two months post-kindling Ž p - 0.01.. There were no significant differences between the one-week and one-month kindled groups Ž p ) 0.05. or between the control group and the two-month kindled group Ž p - 0.05.. These findings suggest that kindling-induced increases in the hilar area are not permanent. Fig. 3 shows representative examples of GFAP immunostained sections in a control rat ŽA., in rats at seven days ŽB., one month post-kindling ŽC. and two months post-kindling ŽD.. These sections clearly show increases in glial cell size in the seven-day and one-month groups. Given that increased GFAP immunostaining is considered to reflect reactive gliosis w13x, hilar GFAP immunostaining was used to quantify reactive gliosis in the present study. Horizontal sections immunostained with GFAP were examined at 400 = magnification using MCID. Hilar GFAP immunostaining was evaluated in a hilar field Ž0.2 mm = 0.48 mm. starting at the hilar end of the CA3rCA4 for each brain section, using MCID’s target detection feature. This feature permits image components to be separated into valid targets and background based on the optical density of the target. Glial cell bodies and processes were regarded as valid targets in the present study. A target
335
acceptance criteria was established using a segmentation range between the upper and lower density thresholds of the target. That is, pixels lying within the segmentation range were regarded as valid targets, whereas pixels lying outside of the range were ignored as background. The segmentation range was set by manually decreasing the thresholding value until a blue overlay display, designating the thresholded area, completely occupied the glial cell bodies and processes Ži.e., the target., but not the background. Fig. 4 shows MCID images of GFAP immunostained hilar fields before ŽA. and after ŽB. density thresholding. The mean proportional area Ži.e., the proportion of the field that is occupied by the target. of GFAP immunostaining was calculated for each hilar field per section. A Ž4 = Ž2 = 6.. ANOVA Žgroup = Žbrain hemisphere= level.. was conducted to evaluate the mean proportional area of hilar GFAP immunostaining. There was a main effect for group: F Ž3,16. s 44.24; p - 0.0001 ŽFig. 5.. Post-hoc Tukey tests showed that the mean proportional area of GFAP immunostaining was significantly increased by approximately 57% at seven days post-kindling, remained elevated at one month post-kindling, but declined back to control levels at two months post-kindling. There were no differences between the seven-day and one-month post-kindling groups Ž p ) 0.05., and there were no differences between the control and the two-month post-kindling groups Ž p ) 0.05.. These findings are similar to those obtained by Hansen et al. Ž1991., who demonstrated that GFAP protein levels remained elevated in most limbic
Fig. 5. Mean proportional area of hilar GFAP immunostaining as a function of treatment condition. A three-way ANOVA with subsequent post-hoc comparisons showed that mean area GFAP immunostaining as a proportion of the total field was significantly elevated at one week post-kindling and remained elevated at one month post-kindling Ž p0.05., but decreased to control levels by two months post-kindling Ž p- 0.05.. Mean proportional area of GFAP immunostaining was not significantly different between either the one-week and the one-month groups Ž p) 0.05., or between the control and the two-month groups Ž p) 0.05.. ) P - 0.05.
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structures at one week post-kindling, but declined to control levels at two months post-kindling w7x. These data suggest that kindling-induced up-regulation of GFAP is transient. The findings in the present study provide the first evidence that kindling-induced changes in the hilar area of the dentate gyrus and kindling-induced changes in GFAP immunostaining levels follow a similar time course. In addition, these findings provide support for the hypothesis that kindling-induced hilar area changes are mediated by kindling-induced glial cell changes. To date, however, the signal which leads to increased GFAP expression following kindling is unclear, and the role of reactive gliosis in kindling remains to be elucidated w11x. Additional research is required not only to define the relevance of glial cell mechanisms in the kindling process, but also to elucidate the role that kindling-induced reactive gliosis plays in mediating kindling-induced hilar area changes in the dentate gyrus.
Acknowledgements
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This work was supported by grants from the Neuroscience Network Centers of Excellence ŽNCE. ŽR.J.R. and M.F.. and the Medical Research Council of Canada ŽMRC. ŽR.J.R. and M.F... B.A. was supported by a studentship from the Savoy Foundation and a supplement from the NCE.
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