42 A
quantified at 11.6 mm. The rationale for choosing these 2.9. Statistical analyses
structures was based on previous research on non-injured brains showing c-Fos expression in these regions after
All statistical analyses were performed using Statview partial and or generalized seizure activity [16].
software Abacus Concepts, 4.51 on a Macintosh com- puter. One- and two-factor analyses of variance ANOVAs
´ 2.7.2. bFGF immunoreactivity
were used followed by Scheffe post hoc tests when Basic FGF positive astrocytes were viewed with a 203
appropriate. The behavioral data are expressed as the group objective on the same imaging equipment used for observ-
mean6standard error S.E. and the immunohistochemical ing c-Fos. Two 50-mm coronal sections 11.60, 11.20
data are reported as difference scores derived by subtract- mm relative to bregma with a 4003300 mm rectangular
ing the mean of the control groups e.g., Non-kindled, ¨
grid superimposed on the frontal cortex areas 1 and 2, Non-kindled, and Naıve from the main groups Stage 0,
lateral to the lesion, the corpus callosum directly under Stage 1, and Lesion-only, respectively. The data were
the cingulum, including the forceps of the minor corpus considered significant when probability values were less
callosum, and the dorsal striatum directly below the than 0.05.
corpus callosum were quantified in the lesioned-hemi- ¨
sphere. In the case of the Naıve group, which did not have a lesion or electrode, the hemisphere for quantification was
3. Results
randomly chosen. Only cells displaying the morphology of reactive astrocytes and exhibiting distinct processes and
3.1. Histology common to all experiments soma [65] were physically counted by an observer blind to
group conditions. An expert neuroanatomist in our depart- Regions of the prefrontal cortex sustaining damage due
ment Dr Eva Fifkova observed several random sections to the AMC lesion included the anterior cingulate and
and agreed with our findings. medial agranular cortices as well as portions of the
prelimbic and infralimbic areas. These regions are con- 2.8. Experimental design
sistently damaged to the same degree following our AMC injury model and have been depicted elsewhere [38].
2.8.1. Experiment 1. AMC lesion1ipsilateral hemisphere Kindling electrode tips were located in the following
kindling nuclei of the amygdala with equal representation among
Thirty-six rats Non-kindled510, Stage 0 single57, groups: central, basolateral, lateral and medial.
Stage 0 multiple59, Stage 1510 underwent AMC lesion and kindling electrode placement in the same hemisphere
3.2. Experiment 1. AMC lesion1ipsilateral hemisphere as described in Section 2.2.
kindling 2.8.2. Experiment 2. AMC lesion1contralateral
3.2.1. Magnitude of asymmetry hemisphere kindling
A repeated measures ANOVA revealed a significant Thirty-eight rats Non-kindled510, Stage 0 single510,
difference among groups F 57.841, P50.0005, a
3,32
Stage 0 multiple59, Stage 159 were used. With the significant difference over days F
538.909, P,
13,416
exception of placing the kindling electrode in the hemi- 0.0001 and a significant group3day interaction F
5
39,416
sphere contralateral to the AMC lesion all procedures were 2.337, P,0.0001. Fig. 1 illustrates that Stage 1 seizures
identical to those described for Expt. 1. elicited during the critical period following a unilateral
AMC lesion significantly impeded functional recovery. ´
2.8.3. Experiment 3. AMC lesion1ipsilateral hemisphere Scheffe comparisons demonstrated that the Stage 1 group
kindling : c-Fos and bFGF immunohistochemistry
differed significantly from the Stage 0 single, Stage 0 Eighteen Lesion only54, Non-kindled54, Stage 055,
multiple, and Non-kindled groups all Ps,0.0001, but Stage 155 of 22 rats underwent surgery as described for
the latter groups were not significantly different from one Expt. 1. The remaining four were not subjected to any of
another all Ps.0.05, not significant n.s.. Further inspec- the surgical or behavioral manipulations and thus served as
tion of Fig. 1 reveals that despite all groups displaying ¨
Naıve controls so that a distinction between basal and similar magnitudes of asymmetry initially Non-kindled5
lesion-induced c-Fos and bFGF could be made. Addition- 4.5560.28,
Stage single54.0060.45,
Stage ally, because the results of Expts. 1 and 2 showed that
multiple54.2760.32, Stage 154.7560.36; P50.5006, functional recovery was not significantly different between
one-factor ANOVA, n.s., Stage 1 animals did not recover the Stage 0 single and Stage 0 multiple groups, we
even after 2 months of behavioral testing. A one-factor evaluated in this experiment only the Stage 0 group
ANOVA on the last day of behavioral testing Day 63 receiving multiple stimulations so that the number of
revealed an asymmetry score of 2.4560.49 for the Stage 1 kindling trials could be kept consistent with the Stage 1
group that was significantly different from the 0.4060.40 group.
of the Non-kindled, the 0.0060.00 of the Stage 0 single,
A .E. Kline et al. Brain Research 880 2000 38 –50
43
Fig. 1. Mean 6S.E. magnitude of asymmetry following a unilateral Fig. 2. Mean 6S.E. magnitude of asymmetry following a unilateral
anteromedial cortex lesion in amygdala-kindled rats. Animals were AMC lesion in amygdala-kindled rats. Animals were stimulated contrala-
stimulated ipsilateral to the lesion either once per day Stage 0 single, teral to the lesion either once per day Stage 0 single, or 1–3 times per
or 1–3 times per day Stage 0 multiple and Stage 1, during the critical day Stage 0 multiple and Stage 1, during the critical period, and once
period, and once a day thereafter until a Stage 5 seizure was attained. per day thereafter until a Stage 5 seizure was attained. There were no
Despite all groups displaying similar asymmetries initially, the group differences in recovery patterns among the groups throughout the
exhibiting a Stage 1 seizure during the critical period remained markedly evaluation period all Ps.0.05. This finding is unlike that seen in Fig. 1
impaired throughout the 63-day neurobehavioral evaluation period. where the initiation of Stage 1 seizures ipsilateral to the lesion sig-
Significantly different from Non-kindled, Stage 0 single, and Stage 0 nificantly impaired recovery. Hash-marks denote the defined critical
´ multiple all Ps,0.0001, Scheffe. Hash-marks denote the defined
period of 12 h to 6 days following injury. critical period of 12 h to 6 days post-injury.
the critical period in the hemisphere contralateral to a and the 0.3860.38 asymmetry score of the Stage 0
unilateral AMC lesion did not impact functional recovery. multiple groups P50.0005. Analysis of the latency in
An ANOVA did not yield a significant difference among seconds to respond to equal-sized adhesive stimuli did not
groups F 51.873, P50.8648, nor did it reveal a
3,34
reveal a significant difference among groups P50.3047, group3day interaction F
50.382, P50.9998. There
39,442
indicating that the difference in recovery rates was not was, however, a significant difference over days as the
attributed to a deficit in general behavioral responsiveness. animals recovered F
567.102, P,0.0001. All
13,442
groups displayed equivalent magnitudes of asymmetry on 3.2.2. Amygdala kindling
the first testing day Non-kindled53.8260.43, Stage 0 As designed, the distinct stimulation parameters utilized
single53.7560.27, Stage
multiple,53.9460.44, for each group within the critical period resulted in the
Stage 154.0560.28 F 50.134, P50.9392, one-factor
3,34
desired kindled seizure stage reliably occurring within the ANOVA; n.s.. Similarly, all groups recovered from som-
first 6 days after lesion. During this same time, the amount atosensory asymmetries by Day 42 post-surgery Non-
of accumulated AD differed only between the Stage 0 kindled50.0060.00, Stage 0 single50.0060.00, Stage 0
single 35.52463.532 s group and the groups receiving multiple50.0060.00, Stage 150.3860.38. This finding
multiple stimulations: Stage 0 multiple 73.7765.2 s is in marked contrast to that seen in Expt. 1 where Stage 1
and Stage 1 89.566.80 s, Ps,0.0001, each vs. Stage 0 seizures elicited in the ipsilateral hemisphere prevented
´ single, Scheffe. These data suggest that neither the
functional recovery for more than 2 months. number of kindling trials nor cumulative AD during the
critical period following an AMC lesion were factors influencing the Stage 1 seizure-mediated neurobehavioral
3.3.2. Amygdala kindling impairment. Instead, the functional consequences of kin-
The distinct stimulation parameters utilized for each dled seizures appear to be related to the degree of
group, again resulted in the desired kindled seizure stage convulsive behavior Stage 0 vs. Stage 1 experienced
occurring within the post-lesion critical period. During this during the critical period.
same time, the accumulated AD was significantly lower in the Stage 0 single group 40.0063.48 s when compared
3.3. Experiment 2. AMC lesion1contralateral to the multiple-stimulation groups: Stage 0 multiple
hemisphere kindling 101.9568.25 s and Stage 1 101.7568.69 s, Ps,
´ 0.0001, each vs. Stage 0 single, Scheffe. This finding is
3.3.1. Magnitude of asymmetry not surprising given that the groups receiving multiple
As illustrated in Fig. 2, Stage 1 seizures elicited during stimulations were given more opportunity to produce AD.
44 A
between the Stage 0 multiple or Stage 1 groups as is in agreement with Expts. 1 and 2 above.
3.4.3. Quantification of c-Fos immunoreactive cells Analysis of c-Fos positive cells expressed in the
piriform cortex revealed a significant difference among groups F
59.928, P,0.0001, which was attributed to
9,32
Stage 1 seizures significantly increasing c-Fos induction in the kindled hemisphere compared to all other groups all
´ Ps,0.05, Scheffe Fig. 3. There were no significant
differences revealed by the other comparisons all Ps. ´
0.05, Scheffe. The mean 6S.E. difference score for each of the main groups in the ipsilateral piriform cortex was
7.7567.84 for the Lesion-only group, 6.25614.69 for the Stage 0, and 142.65643.47 for the Stage 1 group. Seizure-
Fig. 3. Mean 6S.E. difference scores in c-Fos expression in the
induced c-Fos did not change significantly in the contrala-
piriform cortex 2 h after a Stage 0 or Stage 1 amygdala-kindled seizure in
teral piriform cortex from that observed ipsilaterally for
rats with a unilateral AMC lesion. Animals experiencing Stage 1 seizures exhibited marked c-Fos expression ipsilateral, but not contralateral, to the
the Lesion-only and Stage 0 groups 6.5069.52 and
lesion. In contrast, Stage 0 seizures did not induce c-Fos above that of the
5.2567.62, respectively, but was dramatically reduced in
Lesion-only denoted in legend as ‘Lesion’ or Non-kindled groups data
the Stage 1 group 16.7565.93.
not shown. These data demonstrate that Stage 1 seizures propagate from
As shown in Table 1, Stage 1 seizures also significantly
the site of stimulation, but remain confined to the kindled hemisphere.
enhanced c-Fos induction in the ipsilateral perirhinal and
´ Significantly different from all other groups all Ps,0.05, Scheffe.
Horizontal dashed line separates ipsilateral from contralateral.
infralimbic cortices all Ps,0.05. With the exception of the Stage 0 seizure group markedly inducing c-Fos in the
ipsilateral infralimbic cortex P,0.05, there were no 3.4. Experiment 3. AMC lesion1Ipsilateral hemisphere
significant differences among the remaining groups in ´
kindling : c-Fos and bFGF immunohistochemistry
either structure all Ps.0.05, Scheffe. The fact that Stage 1 seizures did not significantly elevate c-Fos induction in
the contralateral structures suggests that this seizure type 3.4.1. Magnitude of asymmetry
remained relatively localized in the ipsilateral hemisphere. Animals were assessed on post-operative days 2 and 5 to
Although both the ipsilateral and contralateral sen- determine if all groups sustained a similar degree of injury
sorimotor cortices expressed more c-Fos following Stage 1 prior to immunohistochemical analyses. Analysis of the
seizures in comparison to the Stage 0 and the Lesion-only data revealed that all groups displayed equivalent mag-
groups, the differences were not statistically significant all nitudes of asymmetry on each testing day Day 2 range5
´ Ps.0.05, Scheffe. Additionally, while the Stage 1 group
3.50–4.60 and Day 5 range52.50–3.75. also induced more c-Fos in the ipsilateral striatum when
compared to the other groups, the variability was suffi- 3.4.2. Amygdala kindling
ciently large to yield no statistical differences Ps.0.05. The distinct kindling parameters utilized resulted in the
Similarly, c-Fos expression was increased in the ipsilateral desired kindled seizure stages being evoked within the 6
CA of the Stage 0 and Stage 1 groups, but presumably
3
´ day critical period, prior to sacrifice. There were no
because of excessive variability, the Scheffe post hoc tests differences, however, in the amount of cumulative AD
did not detect significant differences Ps.0.05, n.s..
Table 1
a
Summary of seizure and lesion-induced c-Fos expression Seizure
Stage 0 I Stage 0 C
Stage 1 I Stage 1 C
Lesion I Lesion C
b
Piriform 6.25614.69
5.256 7.62 142.60643.47
16.756 5.93 7.7567.84
6.5069.52
b
Perirhinal 4.006 4.25
3.256 2.35 76.20628.95
14.956 5.19 20.5065.40
20.5063.95
c
Infralimbic 56.50634.22
9.00612.09 96.50644.17
17.25613.28 17.7567.61
7.5065.06 Sensorimotor
5.506 5.05 6.006 5.47
10.106 4.95 13.756 3.90
0.0562.43 3.5062.93
Striatum 0.756 6.38
25.506 3.82 10.256 5.71
20.056 4.92 0.0563.42
0.2563.07 CA
48.92630.70 6.176 5.29
26.67619.39 6.276 1.96
2.7562.45 2.2561.99
3 a
Quantification of c-Fos immunoreactive cells in the kindled hemisphere ipsilateral to the lesion. The Lesion group was not kindled and did not have an indwelling amygdala electrode. I hemisphere ipsilateral to the lesion; C hemisphere contralateral to the lesion. Values are expressed as difference scores
¨ derived by subtracting the mean of the control groups Non-kindled, Non-kindled, and Naıve from the main groups Stage 0, Stage 1, and Lesion-only,
b c
respectively. Five animals were used in each group. Superscript letters reflect significant differences from all conditions P,0.05, from all conditions except Stage 0 I P,0.05.
A .E. Kline et al. Brain Research 880 2000 38 –50
45
Taken together, the results show that Stage 1 seizures for the Stage 0, and 22.2063.54 for the Stage 1 group. A
´ markedly increased the expression of c-Fos positive cells
Scheffe analysis revealed that both the Stage 0 and Lesion- in the piriform, perirhinal, and infralimbic cortices of the
only groups were significantly different from the Stage 1 kindled hemisphere beyond that of Stage 0 seizures. Stage
group P,0.0001 and P50.0062, respectively, but were 1 seizures also increased c-Fos in the sensorimotor cortex,
not significantly different from each other P50.0691, dorsal striatum, and CA sector of the hippocampus, but to
n.s.. The results show both a lesion-induced expression of
3
a lesser extent than in the aforementioned regions. Thus, bFGF-positive astrocytes that is consistent with other
despite spreading beyond the site of stimulation, Stage 1 studies [22,24,41] and an inhibition of endogenous or
seizures remain relatively confined to the kindled hemi- lesion-induced bFGF-positive astrocytes following Stage
sphere Figs. 3 and 4. 1 seizures Fig. 5. The latter finding is exclusive to our
study and may provide an explanation as to why Stage 1 3.4.4. Quantification of bFGF immunoreactive astrocytes
seizures evoked in the hemisphere ipsilateral to the AMC The expression of bFGF-positive astrocytes in the dorsal
lesion impeded somatosensory recovery. striatum following a unilateral AMC lesion coupled with
As seen in Table 2, lesion-induced expression of bFGF- amygdala-kindled
seizures was
significantly different
positive astrocytes was decreased in the sensorimotor among groups F
522.522, P,0.0001. The mean cortex following Stage 0 and Stage 1 seizures. Indeed,
4,39
6S.E. difference scores of immunoreactive astrocytes bFGF immunoreactivity was significantly different among
´ were 19.0063.94 for the Lesion-only group, 17.3064.38
groups P,0.0001. A Scheffe analysis revealed that none
Fig. 4. Photomicrograph providing a visual corroboration of the data in Fig. 3 showing c-Fos immunoreactivity in the piriform cortex 2 h after amygdala-kindled seizures in AMC lesioned rats. c-Fos expression in animals experiencing Stage 0 seizures C,D was not significantly different from that
of Non-kindled A,B or Lesion-only not depicted groups. Conversely, animals experiencing Stage 1 seizures exhibited marked c-Fos staining ipsilateral E, but not contralateral F, to the lesion. Scale bar5250 mm.
46 A
exhibited fewer bFGF-positive astrocytes than the Non- kindled control group as reflected by the negative scores of
the kindled groups. The Lesion-only group displayed significantly more bFGF immunoreactive astrocytes than
its control group, but not significantly more than either the
´ Stage 0 or Stage 1 groups Ps.0.05, Scheffe, n.s..
4. Discussion
