A .J. Ramos et al. Brain Research 883 2000 1 –14
3
paraformaldehyde and 0.25 v v glutaraldehyde in 0.1 M values into ROD by using the formula: ROD5log 256
phosphate buffer, pH 7.4. Brains were removed and kept in mean gray. The ROD value was chosen to evaluate the
the same cold fixative solution for 2–4 h. After that, brains intensity of S-100b and 5HT immunoreactivities. A back-
were washed three times in cold 0.1 M phosphate buffer, ground parameter was obtained from each section out of
pH 7.4, containing 5 w v sucrose, and left in this the immunolabelled structures, and subtracted from each
washing solution for 18 h at 48C. Coronal and saggital cell ROD before statistically processing values. For the
40-mm thick brain sections were cut using a vibratome. evaluation of 5HT-T, Nf-68 and Nf-200 positive fibers, the
The sections were stored at 2208C in 0.1 M phosphate total area of immunolabelled fibers was related to the total
buffer, pH 7.4, with 25 w v sucrose added as a area of the evaluated field, giving a fiber density parame-
cryoprotector. ter.
2.3. Immunohistochemistry 2.5. Statistics
Brain sections of both PCPA-treated and control groups Four to ten separate immunohistochemical experiments
were simultaneously processed in the free floating state. In were run for each primary antibody. Individual experi-
order to inhibit endogenous peroxidase activity, tissue ments were composed of six to ten tissue sections of each
sections were previously dehydrated, treated with 0.5 animal from each group. Seven to ten fields were measured
v v H O in methanol for 30 min at room temperature and for each brain area in each section of each animal. Inter-
2 2
rehydrated. Free-floating brain sections were blocked for 1 animal differences in each group were not significant.
h with 3 v v normal goat serum in phosphate buffer Values represent the means of experiments performed for
saline PBS. After two rinses in PBS, the sections were each marker, time and brain area. Differences among the
incubated for 48 h at 48C with one of the following means were statistically analyzed using one-way ANOVA
primary antibodies against 5HT, S-100b, Nf-200, Nf-68 or and Student–Newman–Keuls post test. Statistical signifi-
5HT-T diluted 1:8000; 1:500; 1:3000; 1:3000 or 1:1000 cance was set at P,0.05.
v v, respectively. Following five rinses in PBS, sections Differences among the means of control groups C0, C1,
were incubated for 1 h at room temperature with C2 were not significant, and one mean for control group is
biotinylated secondary antibodies diluted 1:100. After shown in the figures.
further washing in PBS, sections were incubated for 1 h with streptavidin–peroxidase complex solution diluted
1:200. After washing again five times in PBS and twice in
3. Results
0.1 M acetate buffer, pH 6 AcB, development of peroxidase activity was carried out with 0.035 w v
3.1. Serotonin 3,39-diaminobenzidine plus 2.5 w v nickel ammonium
sulfate and 0.1 v v H O dissolved in AcB. Following The evaluation of 5HT-IR in the somata of serotoniner-
2 2
the enzymatic incubation step, sections were washed in gic neurons from the mesencephalic DRN is useful to test
AcB three times and once in distilled water. Sections were the effectiveness of PCPA treatment in reducing 5HT
mounted on gelatin-coated slides, dehydrated and cover- levels, based on the sensitivity of this nucleus to PCPA
slipped using Permount for light microscopic observation. [47]. In the T0 group, 5HT-IR was significantly reduced in
All antibodies, as well as the streptavidin complex, were the DRN dropping to 40 of control values P,0.001. In
dissolved in PBS containing 1 v v normal goat serum the T1 group immunoreactivity was partially recovered
and 0.3 v v Triton X-100, pH 7.4. 88 of control and it continued to increase to give the
largest value in the T2 group 172 of control; P,0.001 2.4. Morphometric measurement
Fig. 1. PCPA treatment also reduced 5HT-IR in serotoninergic projecting fibers. Varicose fibers with dense
All measurements were performed on coded slides to branching were detected by anti-5HT antibodies in
ensure objectivity. Mean gray of immunostained glial cells striatum, hippocampus, parietal and frontal cortex in the
and total area of fibers were measured in an Axiophot control groups. Meanwhile 5HT-IR fibers disappeared in
Zeiss light microscope equipped with a video camera on the T0 and T1 groups. 5HT-IR fibers from these regions
line with a Zeiss-Kontron
VIDAS
image analyzer. Images were detected again in the T2 group data not shown.
obtained with the light microscope were transferred to a video camera attached and connected to an interactive
3.2. S-100b immunostaining image analysis system on line. The images were digitized
into an array of 5123512 pixels corresponding to 1403 Intracellular S-100b immunoreactivity was observed in
140 mm 403 primary magnification. The resolution of the astroglial cells of every analyzed brain region in
each pixel was 256 gray levels. Relative optical density control and treated groups. The intensity of intracellular
ROD was obtained after a transformation of mean gray S-100b immunoreactivity was increased in the T0 group in
4 A
increased in the T0 and T1 animals 363, P,0.01 and 331 of controls, P,0.01; respectively. In the T2 group,
S-100b immunoreactivity decreased drastically and re- turned to the control level Figs. 2 and 3A–D.
In the frontal cortex, astrocytes showed an increase of intracellular S-100b immunoreactivity in the T0 group
331 of control; P,0.001. In the T1 group, S-100b immunoreactivity was lower, but it was still significantly
increased over the control values 191 of control; P, 0.05. In the T2 group, the intensity of S-100b immuno-
staining was not significantly different from the control animals Figs. 2 and 4A–D.
Astrocytes from the parietal cortex presented a similar
Fig. 1. Optical density of 5HT-IR neurons in the DRN after PCPA
profile, compared with frontal cortex, showing the maxi-
treatment. Data are expressed in ROD units and represent the means of
mal S-100b intracellular immunoreactivity in the T0
four to seven experiments. Error bars represent the standard deviation of the means. Optical density of 5HT-IR neurons in the DRN is significantly
animals 385 of control; P,0.001 followed by a
reduced in the T0 group, whereas it is increased significantly in the T2
recovering towards control level, being 174 of control
group. , P,0.001 vs. control group, after one-way ANOVA and
P,0.01 in the T1 group and not different from control
Student–Newman–Keuls post-test.
levels in the T2 group Fig. 2. all studied regions, but area-dependent differences in the
3.3. Neurofilaments 200 and 68 kDa response were observed during the recovering T1 and T2
groups. In the striatum, we found important alterations in Nf-
In the striatum, astroglial intracellular S-100b immuno- 200 expression pattern. In the T0 and T1 animals, the
reactivity was significantly increased in the T0 group neurofilaments inside striatal patches striosomes were
261 of control; P,0.001. Although, values obtained observed with abnormal, enlarged and fragmented struc-
for T1 and T2 groups were lower showing a trend to ture. The maximal disruption was observed in the T0 and
recover, they were significantly different from controls T1 groups. In the T2 group alterations were lower, and the
193 of control; P,0.01 and 166 of control; P,0.01, total number of Nf-200 labelled fibers were increased Fig.
respectively. Thus,
S-100b immunostaining
in the
5A–H. Image analysis for this region, performed inside striatum did not return to control level 2 weeks after
the patches, was expressed as area of immunostained ending PCPA treatment Figs. 2 and 3E–H.
neurofilaments per unit area of patches. Thus, these data The analysis of hippocampal astrocytes was focused on
imply an abundance of neurofilaments expression in the the stratum radiatum of CA-1, an area containing basal
striosomes. Data obtained in this way showed a progres- dendrites of the pyramidal neurons. After PCPA treatment,
sive reduction from T0 75.1 of control to T1 59.4 of the intracellular S-100b immunostaining was significantly
control, P,0.05. In the T2 group, a significant recovery
Fig. 2. Optical density of S-100b immunostained glial cells after PCPA treatment. Data are expressed in ROD units and represent the means of four to ten experiments for each region. Error bars represent the standard deviation of the means. The significant increase in the optical density of S-100b
immunostained glial cells in the T0 group is followed by a sharp decrease in both T1 and T2 groups. Striatum is the only region that does not recover the S-100b immunostaining control value in the T2 group. , P,0.05, , P,0.01, , P,0.001 vs. control group after one-way ANOVA and
Student–Newman–Keuls post-test.
A .J. Ramos et al. Brain Research 883 2000 1 –14
5
Fig. 3. Above: photographs show S-100b immunostaining in the hippocampus, CA-1 area of stratum radiatum. A control; B 1 day after ending PCPA treatment T0; C 7 days after ending PCPA treatment T1; D 14 days after PCPA treatment T2. Bar523.5 mm. Below: S-100b immunostaining in
the striatum. E control; F 1 day after ending PCPA treatment T0; G 7 days after ending PCPA treatment T1; H 14 days after PCPA treatment T2. Bar523.5 mm.
was observed 96.8 of control, being significantly control level 91.5 of control being significantly differ-
different from T1 P,0.01 vs. T1 Fig. 6. The Nf-68 ent from the T0 group P,0.001 vs. T0. In the T2 group,
expression in the striatal patches also showed a decrease in the number of Nf-200 positive dendrites was greater than
the T0 group 67.5 of control, P,0.01, followed by an in the T1 group, but they were abnormal compared with
increase in the T1 group 85.5 of control; and a further the paired controls Fig. 8A–D. Image analysis confirmed
increase to reach a value of 130.7 of control P,0.01 in the increased number of Nf-200 labelled fibers showing a
the T2 group Figs. 7 and 9E–H. value significantly higher than control 137.1 of control,
In the hippocampus, Nf-200 expression was decreased P,0.05 Fig. 6. The profile of Nf-68 expression in the
in the stratum radiatum of treated animals. In the T0 group, hippocampal areas was altered after the PCPA treatment,
the basal dendrites of pyramidal neurons, as seen by being reduced in the T0 animals, but showing a continuous
Nf-200 immunoreactivity, were shorter and thicker than increase from the T1 to T2 groups. Moreover, in the T2
those from control groups. Also a reduction in the total group Nf-68 expression was increased in CA-1, where it
number of Nf-200 labelled structures was observed Fig. was normally very low. Image analysis focused on the
8A–D. Image analysis, focused in the stratum radiatum of stratum radiatum of CA-1 clearly showed the increased
CA-1 showed a significant reduction in Nf-200 labelled expression of Nf-68 in the T2 group 745.3 of control,
structures 47.0 of control P,0.001. In the T1 group, P,0.001 Fig. 7.
Nf-200 immunoreactive profiles began to recover towards The pattern of Nf-200 expression in the frontal cortex
6 A
Fig. 4. S-100b immunostaining in the frontal cortex. A control; B 1 day after ending PCPA treatment T0; C 7 days after ending PCPA treatment T1; D 14 days after PCPA treatment T2. Arrows show typical images of S-100b immunolabelled astrocytes. Bar522.2 mm.
Fig. 5. Above: immunostaining for 200 KDa neurofilaments Nf-200 in the striatum. A control; B 1 day after ending PCPA treatment T0; C 7 days after ending PCPA treatment T1; D 14 days after PCPA treatment T2. Note the Nf-200 alterations in these low magnification photographs. Bar547
mm. Below: the same region with higher magnification to show morphological alterations in detail. E control; F 1 day after ending PCPA treatment T0 the inset shows a higher magnification of the typical Nf-200 altered morphology found in this model arrow; G 7 days after ending PCPA treatment T1;
H 14 days after PCPA treatment T2. Bar526.7 mm.
A .J. Ramos et al. Brain Research 883 2000 1 –14
7
2
Fig. 6. Area of Nf-200 immunoreactive fibers after PCPA treatment. Data are expressed as area of Nf-200 immunolabelled fibers per mm of tissue and represent the means of six to seven experiments for each region. Error bars represent the standard deviation of the means. The significant decrease in the
striatal for T1 group, hippocampal and cortical for T0 group Nf-200 immunolabelled fibers is followed by a significant increase in the T2 group. , P,0.05, , P,0.01 and , P,0.001 vs. control, after one-way ANOVA and Student–Newman–Keuls post-test. †, For the striatum, values are
2
expressed as area per mm of patches striosomes and multiplied by 0.1 to show them in the same scale as the other brain regions.
showed alterations over time. The total quantity of im- Nf-68 labelled fibers in the T0 group 45.7 of control,
munocytochemically stained Nf-200 was reduced in the T0 P,0.01. In the T1 group, Nf-68 immunoreactivity began
group. The reduction was observed in the thin network of to increase towards the control level 65.7 of control to
cortical dendrites from pyramidal neurons in cortical layers reach control values in the T2 group 98.4 of control,
I, II and III. Image analysis, focused in the cortical layer being significantly different from T0 group P,0.01 Fig.
III showed a reduction in Nf-200 immunostaining 85 of 7.
control, but it was not statistically significant. In the T1 Nf-200 immunolabelling in the parietal cortex showed a
and T2 groups, Nf-200 labelled structures in the cortical pattern that shares some observations with the frontal
layer III were significantly increased 139 and 152, cortex. In the T0 group the Nf-200 density was sig-
respectively, of control, P,0.01 for both in the T2 group nificantly reduced in the network of dentrites from pyrami-
Fig. 6. In the T0 group Nf-68 immunolabelling was dal neurons. Image analysis, focused in the cortical layer
reduced in dendrites and soma of pyramidal neurons from III confirmed this observation showing a value of 62.4 of
cortical layers III and V. Image analysis, again focused on control P,0.001, being a more important reduction than
the cortical layer III showed a significant reduction of in frontal cortex. In the T1 group, an increased number of
2
Fig. 7. Area of Nf-68 immunoreactive fibers after PCPA treatment. Data are expressed as area of Nf-68 immunolabelled fibers per mm of tissue and represent the means of five to eight experiments for each brain region. Error bars represent the standard deviation of the means. The significant decrease in
the striatal and cortical Nf-200 immunolabelled fibers in the T0 group is followed by a significant increase in the T2 group. Hippocampal Nf-68 immunolabelled fibers in the stratum radiatum of CA-1 also increases in the T2 group. , P,0.01, , P,0.001 vs. control, after one-way ANOVA and
2
Student–Newman–Keuls post-test. †, For the striatum, values are expressed as area per mm of patches striosomes and multiplied by 0.1 to show them in the same scale as the other brain regions.
8 A
Fig. 8. Immunostaining for 200 kDa neurofilaments Nf-200 in the hippocampus, CA-1 area of stratum radiatum. A control; B 1 day after ending PCPA treatment T0; C 7 days after ending PCPA treatment T1; D 14 days after PCPA treatment T2. Note the decreased number of Nf-200 positive
fibers in the T0 group, and the increase in the T2 group. Bar552.2 mm.
Nf-200 immunolabelled dendrites was observed 91.8 of 5HT-T fibers were increased presenting a value similar to
control, being significantly different from T0 P,0.05 vs. control groups 96.5 of control. The area of 5HT-T
T0 group. In the T2 group, Nf-200 immunoreactivity labelled fibers in the T2 group was not significantly
continued increasing to reach a value of 106 of control, different from control groups 101.0 of control Fig.
giving a value significantly different from T0 group P, 10.
0.001 Fig. 6. Nf-68 expression was significantly reduced In the T0 group 5HT-T positive fibers from the frontal
in the dendrites and somata of pyramidal neurons in the T0 cortex were not different from those of control groups
group 49.8 of control, P,0.01. In the T1 group the 99.7 of control. However, a significant increase in the
density of Nf-68 immunolabelled structures began to 5HT-T positive fibers was demonstrated in the T1 146.8
recover towards the control level reaching 83.3 of of control; P,0.001 and T2 groups 131.2 of control;
control groups, being significantly different from T0 P, P,0.001 Figs. 10 and 11E–H.
0.01 vs. T0. Nf-68 immunoreactivity continued increasing The area covered by 5HT-T labelled fibers in the
in the T2 group reaching a value of 175.1 of control parietal cortex was lower compared with the frontal cortex.
P,0.001 Figs. 7 and 9A–D. Quantification of 5HT-T immunostained fibers showed an
increase in the T0 and T1 groups 144.4 and 216.2, 3.4. 5HT-transporter 5HT-T
respectively; only in the T1 group did it prove to be significantly different from controls P,0.001. In the T2
5HT-T is expressed in every 5HT fiber, it is useful to group, the area of 5HT-T labelled fibers dropped to a value
follow 5HT fibers when they are depleted of neurotrans- not significantly different from the controls Fig. 10.
mitter, because they are not detected by anti-5HT anti- bodies. 5HT-T expression was observed in all studied
areas as thin fibers with a dense branching. Quantitative
4. Discussion
