2 A
oxide synthase NOS activity [48] and reduces MAP-2 neurite growth see [20,23] for review and 200-kDa
and synaptophysin immunoreactivities [5]. In addition, neurofilaments Nf-200 which are characteristic of mature
5HT is involved in the regulation of hippocampal and axons see [40] for review. Specific morphology of 5HT
subventricular neurogenesis in the adult brain [6]. 5HT’s fibers has been studied by 5HT transporter 5HT-T
role in the development and plasticity of CNS seems to be immunostaining. Recovery of 5HT has been followed by
mediated by the stimulation of glial 5HT receptors that
5HT immunoreactivity 5HT-IR in the dorsal raphe
1A
cause S-100b protein release [51]. S-100b is a growth nucleus DRN and innervation areas.
factor that derives from glial cells. S-100b has important effects on axonal growth of 5HT neurons [2,3,33], partici-
pates in neurite extension in 5HT innervated areas [24,39],
2. Materials and methods
stabilizes neuronal cytoskeleton [18] and regulates GAP-43 phosphorylation [32].
PCPA methyl ester, mouse monoclonal anti-S-100b Neurofilaments are the major cytoskeletal constituents of
protein, mouse monoclonal anti-Nf-200, mouse monoclo- neurons. They are intrinsic determinants of axonal caliber
nal anti-Nf-68, secondary biotinylated antibodies and [19] and their dynamic remodeling is necessary for axonal
streptavidin complex
used for
immunohistochemistry growth and guidance. By regulating axonal caliber, neuro-
studies were purchased from Sigma. Monoclonal anti 5HT- filaments proteins can affect both axonal transport and
T serum was purchased from Chemicon. Polyclonal rabbit neuronal function [40]. Down-regulation of neurofilaments
antiserum against 5HT was produced and characterized in expression and the presence of altered neurofilaments are
our laboratory [7]. All chemical substances were of common in several human neurodegenerative diseases see
analytical grade. for review [23]. Recent evidence coming from transgenic
mouse shows that disorganized neurofilaments can lead to 2.1. Treatment
neuronal degeneration and death [23]. Conversely, trans- genic mice over expressing S-100b show significant
Thirty adult male Wistar rats weighing 250–300 g were alterations in neuronal cytoskeleton [53].
used. The model of PCPA treatment was modified from a There is evidence of defects in the 5HT system in
previous report [47]. Fifteen rats were treated with PCPA Alzheimer’s disease [8,17,55]. Brains from patients with
and another fifteen rats with saline. The sterile solution Alzheimer’s disease present increased level of S-100b
containing PCPA was injected intraperitoneally i.p. for 14 protein [14], altered neurofilament expression [23], glial
days. Individual PCPA doses were 100 mg kg day, in- reaction and increased NOS activity [42]. Moreover, a high
jection volumes ranged from 0.2 to 0.3 ml. All doses were S-100b level seems to be involved in the pathogenesis of
injected in the morning between 9:00 and 10:00 a.m. The Alzheimer’s disease [15,31,46]. Considering our previous
amount of PCPA injected was one-third of that which findings of a glial reaction [43,47] and increased NOS
inhibits 33 of the brain protein synthesis when it is activity [48] after 5HT depletion, as well as the relation-
administered intravenously [30]. Animals were divided ship between 5HT levels and S-100b, it is interesting to
into three experimental groups T0, T1, T2 and three study the consequences of an experimental lack of 5HT on
control groups C0, C1, C2 of five animals each. Ex- neuronal cytoskeleton and glial S-100b level in the adult
perimental groups were treated with PCPA, control groups brain.
received the same volume of sterile saline solution and In spite of the wide range of pharmacological models
were kept in the same environment as those treated with available to reach an effective 5HT depletion, we have
PCPA 12 h light–dark cycle, controlled humidity and chosen to treat with an inhibitor of tryptophan hydroxy-
temperature, free access to standard rat food and water. lase, parachlorophenylalanine PCPA [25] that produce a
The animal care for this experimental protocol was in significant depletion of brain 5HT [1,25,44,49] leaving the
accordance with the NIH guidelines for the Care and Use 5HT innervation intact [10]. Other common serotoninergic
of Laboratory Animals and the principles presented in the drugs
i.e. substituted
amphetamines, dihydroxytryp-
Guidelines for the Use of Animals in Neuroscience Re- tamines also produce significant 5HT depletion, but they
search by the Society for Neuroscience. are potent neurotoxins for 5HT neurons [10,37,38,45].
In this work we report the study of the neuronal and 2.2. Fixation
glial response after a PCPA induced 5HT depletion in adult rats. The relationship between glial S-100b level and
At 1 day T0 and C0, 7 days T1 and C1 or 14 days morphological alterations on neurons in dense 5HT inner-
T2 and C2 after the last injection, between 9:00 and vated areas has been studied at different times after the
10:00 a.m., animals were deeply anaesthetized with 300 PCPA treatment. Two neurofilaments have been used to
mg kg of chloral hydrate. They were perfused through the follow neuronal cytoskeletal alterations: 68-kDa neurofila-
left ventricle, initially with a cold saline solution con- ments Nf-68, one of the triplet neurofilament proteins
taining 0.05 w v NaNO plus 50 I.U. of heparin and
2
which expression is increased during maturation and subsequently with a fixative solution containing 4
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
