Brain Research 883 (2000) 1–14www.elsevier.com/ locate /bres

Research report

Neuroglial interactions in a model of para-chlorophenylalanine-induced serotonin depletion

´ ´Alberto Javier Ramos, Patricia Tagliaferro, Ester Marıa Lopez, Jorge Pecci Saavedra,*Alicia Brusco

´Instituto de Biologıa Celular y Neurociencia ‘‘Prof. Eduardo De Robertis’’, Facultad de Medicina, Universidad de Buenos Aires,Paraguay 2155 (1121), Buenos Aires, Argentina

Accepted 15 August 2000

Abstract

Serotonin (5HT) is involved in the development and plasticity of the CNS through the release of S-100b, a glial trophic factor whichstabilizes synapses and neuronal cytoskeleton and promotes neuronal development. S-100b is released from glial cells after activation ofglial 5HT receptors. We present in this paper the effects upon neurons and glia of a 5HT depletion induced by 14 days of treatment with1A

para-chlorophenylalanine (PCPA) in adult rats. S-100b, 5HT, 5HT-transporter (5HT-T) and neurofilaments (Nf-200 and Nf-68)expressions were studied by immunohistochemistry and image analysis in striatum, hippocampus, parietal and frontal cortex. Immediatelyafter ending PCPA treatment we found increased intracellular S-100b immunoreactivity in glial cells, reduced 5HT immunolabelling,reduced density of 5HT-T, Nf-200 and Nf-68 fibers and morphological alterations in neuronal cytoskeleton. One week after PCPAtreatment S-100b immunoreactivity decreased towards control levels, 5HT was normalized in dorsal raphe nucleus, but not in innervationareas; 5HT-T, Nf-200 and Nf-68 fiber densities increased but some neuronal cytoskeletal alterations were still present in striatum. Twoweeks after PCPA treatment S-100b had returned to control levels in most studied regions; 5HT immunoreactivity was normalized,meanwhile 5HT-T, Nf-200 and Nf-68 fiber densities increased reaching values over the control level. We propose that S-100b could beaccumulated in glial cells during the 5HT depletion period, to be released once 5HT levels have recovered. Neuronal cytoskeletalalterations and reduced fiber density may be the expression of decreased extracellular availability of S-100b. Conversely, increased5HT-T, Nf-200 and Nf-68 expressions, once S-100b is normalized, may be the biological response to the growth factor release. 2000Elsevier Science B.V. All rights reserved.

Theme: Development and regeneration

Topic: Neurotrophic factors: biological effects

Keywords: Serotonin; Parachlorophenylalanine; Neurotrophic factor; S-100b; Neurofilament; Serotonin transporter

1. Introduction 5HT role as a differentiation signal in the development ofthe central nervous system (CNS). This hypothesis is

Serotoninergic neurons located in the brainstem project supported by several facts: (i) 5HT is the classicalto cortical and subcortical brain regions through a network neurotransmitter expressed in the early brain development,of fibers with dense branching. Terminal fibers containing prior to finishing synaptogenesis and before reaching theserotonin (5HT) present a broad distribution at synaptic as complete stabilization of neurotransmission systems and itwell as non-synaptic sites, raising the hypothesis of seems to promote neuronal growth [26,28]; (ii) neuronalanother role for this amine, besides neurotransmission. differentiation in specific regions correlates with the arrival

The early expression of 5HT in the brain development of 5HT axons [27]; (iii) a depletion of 5HT in pregnantwas the starting evidence [26,27] for the hypothesis of the rats [26] or during the synaptogenesis [34] leads to

decreased brain development.In the adult brain, 5HT plays a role in neuronal and glial*Corresponding author. Correspondence address: Rivera Indarte 132

plasticity. It has been reported that a 5HT depletion1ro ‘‘A’’, (1406), Buenos Aires, Argentina. Fax: 154-11-4941-5618.E-mail address: hbrusco@fmed.uba.ar (A. Brusco). produces significant glial reaction [11,47], increases nitric

0006-8993/00/$ – see front matter 2000 Elsevier Science B.V. All rights reserved.PI I : S0006-8993( 00 )02862-6

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oxide synthase (NOS) activity [48] and reduces MAP-2 neurite growth (see [20,23] for review) and 200-kDaand synaptophysin immunoreactivities [5]. In addition, neurofilaments (Nf-200) which are characteristic of mature5HT is involved in the regulation of hippocampal and axons (see [40] for review). Specific morphology of 5HTsubventricular 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 bymediated by the stimulation of glial 5HT receptors that 5HT immunoreactivity (5HT-IR) in the dorsal raphe1A

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 importanteffects on axonal growth of 5HT neurons [2,3,33], partici-pates in neurite extension in 5HT innervated areas [24,39], 2. Materials and methodsstabilizes neuronal cytoskeleton [18] and regulates GAP-43phosphorylation [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 immunohistochemistrygrowth 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 rabbitneuronal function [40]. Down-regulation of neurofilaments antiserum against 5HT was produced and characterized inexpression and the presence of altered neurofilaments are our laboratory [7]. All chemical substances were ofcommon in several human neurodegenerative diseases (see analytical grade.for review [23]). Recent evidence coming from transgenicmouse shows that disorganized neurofilaments can lead to 2.1. Treatmentneuronal degeneration and death [23]. Conversely, trans-genic mice over expressing S-100b show significant Thirty adult male Wistar rats weighing 250–300 g werealterations 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 PCPAAlzheimer’s disease [8,17,55]. Brains from patients with and another fifteen rats with saline. The sterile solutionAlzheimer’s disease present increased level of S-100b containing PCPA was injected intraperitoneally (i.p.) for 14protein [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 wereS-100b level seems to be involved in the pathogenesis of injected in the morning between 9:00 and 10:00 a.m. TheAlzheimer’s disease [15,31,46]. Considering our previous amount of PCPA injected was one-third of that whichfindings of a glial reaction [43,47] and increased NOS inhibits 33% of the brain protein synthesis when it isactivity [48] after 5HT depletion, as well as the relation- administered intravenously [30]. Animals were dividedship between 5HT levels and S-100b, it is interesting to into three experimental groups (T0, T1, T2) and threestudy 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 groupsbrain. 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 withavailable to reach an effective 5HT depletion, we have PCPA (12 h light–dark cycle, controlled humidity andchosen 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 insignificant depletion of brain 5HT [1,25,44,49] leaving the accordance with the NIH guidelines for the Care and Use5HT innervation intact [10]. Other common serotoninergic of Laboratory Animals and the principles presented in thedrugs (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. Fixationglial response after a PCPA induced 5HT depletion in adultrats. The relationship between glial S-100b level and At 1 day (T0 and C0), 7 days (T1 and C1) or 14 daysmorphological alterations on neurons in dense 5HT inner- (T2 and C2) after the last injection, between 9:00 andvated areas has been studied at different times after the 10:00 a.m., animals were deeply anaesthetized with 300PCPA treatment. Two neurofilaments have been used to mg/kg of chloral hydrate. They were perfused through thefollow 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 and2

which expression is increased during maturation and subsequently with a fixative solution containing 4%

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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 thethe 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 ofpH 7.4, containing 5% w/v sucrose, and left in this the immunolabelled structures, and subtracted from eachwashing solution for 18 h at 48C. Coronal and saggital cell ROD before statistically processing values. For the40-mm thick brain sections were cut using a vibratome. evaluation of 5HT-T, Nf-68 and Nf-200 positive fibers, theThe sections were stored at 2208C in 0.1 M phosphate total area of immunolabelled fibers was related to the totalbuffer, 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 experimentswere 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 eachsections were previously dehydrated, treated with 0.5% animal from each group. Seven to ten fields were measuredv/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 forsaline (PBS). After two rinses in PBS, the sections were each marker, time and brain area. Differences among theincubated for 48 h at 48C with one of the following means were statistically analyzed using one-way ANOVAprimary 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 isbiotinylated secondary antibodies diluted 1:100. After shown in the figures.further washing in PBS, sections were incubated for 1 hwith streptavidin–peroxidase complex solution diluted1:200. After washing again five times in PBS and twice in 3. Results0.1 M acetate buffer, pH 6 (AcB), development ofperoxidase activity was carried out with 0.035% w/v 3.1. Serotonin3,39-diaminobenzidine plus 2.5% w/v nickel ammoniumsulfate 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 testAcB three times and once in distilled water. Sections were the effectiveness of PCPA treatment in reducing 5HTmounted on gelatin-coated slides, dehydrated and cover- levels, based on the sensitivity of this nucleus to PCPAslipped 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). Indissolved in PBS containing 1% v/v normal goat serum the T1 group immunoreactivity was partially recoveredand 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 denseAll 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 theand total area of fibers were measured in an Axiophot control groups. Meanwhile 5HT-IR fibers disappeared inZeiss light microscope equipped with a video camera on the T0 and T1 groups. 5HT-IR fibers from these regionsline 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 avideo camera attached and connected to an interactive 3.2. S-100b immunostainingimage analysis system on line. The images were digitizedinto an array of 5123512 pixels corresponding to 1403 Intracellular S-100b immunoreactivity was observed in140 mm (403 primary magnification). The resolution of the astroglial cells of every analyzed brain region ineach 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

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increased in the T0 and T1 animals (363%, P,0.01 and331% 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 ofintracellular 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 significantlyincreased 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 controlanimals (Figs. 2 and 4A–D).

Astrocytes from the parietal cortex presented a similarFig. 1. Optical density of 5HT-IR neurons in the DRN after PCPAprofile, compared with frontal cortex, showing the maxi-treatment. Data are expressed in ROD units and represent the means ofmal S-100b intracellular immunoreactivity in the T0four 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 areduced in the T0 group, whereas it is increased significantly in the T2 recovering towards control level, being 174% of controlgroup. ***, P,0.001 vs. control group, after one-way ANOVA and

(P,0.01) in the T1 group and not different from controlStudent–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 T2groups). 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, thereactivity 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 andrecover, 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 insidestriatum did not return to control level 2 weeks after the patches, was expressed as area of immunostainedending 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 thethe 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% ofthe 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 tenexperiments 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 theS-100b immunostaining control value in the T2 group. *, P,0.05, **, P,0.01, ***, P,0.001 vs. control group after one-way ANOVA andStudent–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 PCPAtreatment (T0); (C) 7 days after ending PCPA treatment (T1); (D) 14 days after PCPA treatment (T2). Bar523.5 mm. Below: S-100b immunostaining inthe 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 thanthe T0 group (67.5% of control, P,0.01), followed by an in the T1 group, but they were abnormal compared withincrease in the T1 group (85.5% of control); and a further the paired controls (Fig. 8A–D). Image analysis confirmedincrease to reach a value of 130.7% of control (P,0.01) in the increased number of Nf-200 labelled fibers showing athe 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 thein 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 continuousNf-200 immunoreactivity, were shorter and thicker than increase from the T1 to T2 groups. Moreover, in the T2those from control groups. Also a reduction in the total group Nf-68 expression was increased in CA-1, where itnumber of Nf-200 labelled structures was observed (Fig. was normally very low. Image analysis focused on the8A–D). Image analysis, focused in the stratum radiatum of stratum radiatum of CA-1 clearly showed the increasedCA-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

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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 daysafter ending PCPA treatment (T1); (D) 14 days after PCPA treatment (T2). Note the Nf-200 alterations in these low magnification photographs. Bar547mm. 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

2Fig. 6. Area of Nf-200 immunoreactive fibers after PCPA treatment. Data are expressed as area of Nf-200 immunolabelled fibers per mm of tissue andrepresent the means of six to seven experiments for each region. Error bars represent the standard deviation of the means. The significant decrease in thestriatal (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

2expressed 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 begangroup. The reduction was observed in the thin network of to increase towards the control level (65.7% of control) tocortical 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 aand T2 groups, Nf-200 labelled structures in the cortical pattern that shares some observations with the frontallayer 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 layerreduced in dendrites and soma of pyramidal neurons from III confirmed this observation showing a value of 62.4% ofcortical layers III and V. Image analysis, again focused on control (P,0.001), being a more important reduction thanthe cortical layer III showed a significant reduction of in frontal cortex. In the T1 group, an increased number of

2Fig. 7. Area of Nf-68 immunoreactive fibers after PCPA treatment. Data are expressed as area of Nf-68 immunolabelled fibers per mm of tissue andrepresent the means of five to eight experiments for each brain region. Error bars represent the standard deviation of the means. The significant decrease inthe striatal and cortical Nf-200 immunolabelled fibers in the T0 group is followed by a significant increase in the T2 group. Hippocampal Nf-68immunolabelled 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

2Student–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 inthe same scale as the other brain regions.

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Fig. 8. Immunostaining for 200 kDa neurofilaments (Nf-200) in the hippocampus, CA-1 area of stratum radiatum. (A) control; (B) 1 day after endingPCPA treatment (T0); (C) 7 days after ending PCPA treatment (T1); (D) 14 days after PCPA treatment (T2). Note the decreased number of Nf-200 positivefibers 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 tocontrol), being significantly different from T0 (P,0.05 vs. control groups (96.5% of control). The area of 5HT-TT0 group). In the T2 group, Nf-200 immunoreactivity labelled fibers in the T2 group was not significantlycontinued 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 frontalin the dendrites and somata of pyramidal neurons in the T0 cortex were not different from those of control groupsgroup (49.8% of control, P,0.01). In the T1 group the (99.7% of control). However, a significant increase in thedensity 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 thein 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 T25HT-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 studiedareas as thin fibers with a dense branching. Quantitative 4. Discussionstudies allowed the evaluation of the fiber density, ex-pressed as the area of 5HT-T immunoreactive fibers per PCPA treatment is very effective at producing importantunit area of tissue. 5HT depletion in brain 5HT innervation areas

5HT-T positive fibers in the striatum were observed in [1,13,25,47,49]. Some side effects in brain protein syn-the matrix, sparing the patches. The image analysis was thesis have been described for high endovenous PCPAfocused on the matrix where dense 5HT innervation is doses [29,30]. Considering this, we used a lower dose bypresent. The area of striatal fibers expressing 5HT-T was the i.p. route for our model that does not produce the samesignificantly reduced in the T0 group (66.4% of control; plasmatic PCPA level [12] that could saturate brain aro-P,0.001) followed by a sharp increase in the T1 (131.3% matic amino acid carriers. We have chosen PCPA becauseof control, P,0.01) and T2 groups (169.7% of control; it produces an important 5HT depletion without beingP,0.001). (Figs. 10 and 11A–D). neurotoxic to 5HT terminal fibers [10]. In a previous paper

Hippocampal 5HT-T labelled fibers were reduced in the we showed that neuronal 5HT-IR in the mesencephalicT0 group (70.0% of control; P,0.001). In the T1 group, DRN is useful to follow 5HT levels in the CNS after PCPA

A.J. Ramos et al. / Brain Research 883 (2000) 1 –14 9

Fig. 9. Above: immunostaining for 68 kDa neurofilaments (Nf-68) in the parietal cortex. (A) control; (B) 1 day after ending PCPA treatment (T0); (C) 7days after ending PCPA treatment (T1); (D) 14 days after PCPA treatment (T2). Note that Nf-68 immunostaining decreased significantly in the dendritesand soma (arrow) of cortical pyramidal neurons in the T0 group. Bar522.2 mm. Below: immunostaining for Nf-68 in the striatum. (E) control; (F) 1 dayafter ending PCPA treatment (T0); (G) 7 days after ending PCPA treatment (T1); (H) 14 days after PCPA treatment (T2). Decreased Nf-68 immunostainingin the T0 group is followed by an increase in the T1 and T2 groups. Bar541.2 mm.

treatment [47]. Astroglial reaction detected in this model groups, they were observed again in the T2 group. Theof PCPA induced 5HT depletion reaches a peak 1 week recovery delay observed in 5HT-IR fibers is in accordanceafter the PCPA treatment, and seems to be completely with previous reports [1,49]. Biochemical reports havereversible [43]. indicated that the 5HT level is recovered in 12–15 days

In this paper we studied the time-course events of glial [25] after PCPA treatment, probably involving an up-and neuronal response after PCPA treatment. The treatment regulation of TrpOH gene expression [41]. In spite of theeffectiveness was analyzed by the quantification of 5HT-IR absence of 5HT-IR fibers in innervation areas in the T0in the DRN and the observation of 5HT-IR fibers in group, 5HT-T immunoreactivity has demonstrated thatinnervation areas. The 5HT-IR in the somata of DRN serotoninergic fibers are present, but depleted of 5HT. Thisneurons was significantly reduced by PCPA treatment. evidence confirms previous results which demonstratedOnce the treatment ended, 5HT-IR rapidly recovered that PCPA is not neurotoxic for serotoninergic projectionstowards the control level. In the T2 group, 5HT-IR was [10].greater than in the control group, probably reflecting the Glial response was followed by the analysis of intracel-demonstrated up-regulation of tryptophan hydroxylase lular S-100b immunoreactivity in astroglia. Under the(TrpOH) gene transcription [41]. Serotoninergic fibers standardized conditions used in our experiments, theanalyzed by 5HT-IR were not detectable in the T0 and T1 intensity of S-100b immunoreactivity can be related to the

10 A.J. Ramos et al. / Brain Research 883 (2000) 1 –14

2Fig. 10. Area of 5HT-T immunolabelled fibers after PCPA treatment. Data are expressed as area of 5HT-T immunolabelled fibers per mm of tissue andrepresent the means of five to nine experiments for each region. Error bars represent the standard deviation of the means. The significant decrease in thestriatal and hippocampal 5HT-T immunolabelled fibers is followed by a significant increase in the T1 and T2 groups. Parietal and frontal cortex alsoshowed a significant increase in the 5HT-T fibers in the T1 (parietal and frontal cortex) and T2 groups (only frontal cortex). *, P,0.05, **, P,0.01, ***,P,0.001 vs. control, after one-way ANOVA and Student–Newman–Keuls post-test.

Fig. 11. Above: immunostaining for 5HT-Transporter (5HT-T) in the striatum. (A) control; (B) 1 day after ending PCPA treatment (T0); (C) 7 days afterending PCPA treatment (T1); (D) 14 days after PCPA treatment (T2). Bar521.5 mm. Below: immunostaining for 5HT-T in the frontal cortex. (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). Bar540 mm.

A.J. Ramos et al. / Brain Research 883 (2000) 1 –14 11

S-100b protein level in astroglial cells. Intracellular S- creased caliber, fragmentation and loss striosomal organi-100b immunostaining is increased after PCPA treatment. zation. These alterations are important in the T0 and T1In fact, this result is not totally in accordance with those groups, but are minimal in the T2 group. The decreasedfound in other 5HT-depletion models [16]. Considering Nf-200 positive fibers in hippocampal CA-1 area, wherethat the activation of glial 5HT receptors (by 5HT or basal dentrites from pyramidal neurons are located, have1A

another agonist) leads to the release of S-100b from been also described by other authors who found decreasedastrocytes [51,52,54], we propose that low 5HT levels immunostaining for the dendritic marker MAP-2 in anothercould reduce such a release giving an accumulation of model of 5HT depletion [5]. Our results showed thatsynthesized S-100b within the astrocytes. However, it neurofilaments expression is reduced and Nf-200 mor-cannot be discounted that up-regulation of S-100b protein phological alterations are important when intracellular S-synthesis might contribute to the observed increase of the 100b immunostaining is maximal, probably reflecting theintracellular S-100b immunostaining. The most important reduced extracellular availability of this growth factor.intracellular S-100b accumulation in astroglial cells was Interestingly, in brains of patients with Alzheimer’s dis-observed when 5HT-IR was minimal, in the T0 group. ease, neurofilament alterations are found together withDuring the recovery of the 5HT levels, S-100b immuno- increased S-100b immunostaining in astrocytes [14,15].reactivity decreased dramatically. Two weeks after ending The increased Nf-68 and Nf-200 expressions in somethe treatment (T2 group) both cortex and hippocampus regions in the T2 group might indicate a sproutinghave recovered the normal S-100b immunostaining, phenomena after S-100b level are normalized. The ob-whereas the striatum still shows more intense S-100b served sprouting could be an in vivo effect of releasedimmunoreactivity. Considering the recovery of the 5HT S-100b.level after PCPA treatment, it could be hypothesized that The study of 5HT fibers was performed following theaccumulated S-100b is released from glial cells when the 5HT-T immunoreactivity. The reduction in striatal and5HT level recovers. The expression of this event may be hippocampal 5HT-T positive fibers in the T0 group was anthe reduction of the intracellular S-100b immunostaining. unexpected result, because PCPA is not neurotoxic forInterestingly, maximal S-100b immunostaining, which 5HT projections [10]. Considering our hypothesis ofoccurred 1 day after the treatment, did not seem to be reduced extracellular availability of S-100b due to the lackcoincident with the maximal astroglial reaction, that we of 5HT stimulation, we propose that the reduction in the1A

demonstrated by glial fibrillary acidic protein (GFAP) 5HT-T fibers might be secondary to the reduced extracellu-immunostaining 1 week after the PCPA treatment [43]. lar level of the growth factor. It has been proposed that theMoreover, glial morphology was not totally recovered until release of glial S-100b could cause regeneration or sprout-35 days post-treatment [unpublished observations]. ing of neuronal terminals [4,52]. This hypothesis may be

It is well known that serotoninergic raphe nuclei have supported by the similar profile of neurofilament expres-different sensitivities to PCPA and other serotoninergic sion and the increase in 5HT-T fibers found in the T1 andtoxins, being the DRN most affected by PCPA and other T2 groups. Moreover, considering that S-100b stabilizesserotoninergic toxins, whereas the median raphe is less the neuronal cytoskeleton [18], we may speculate thatsensitive [35,36,47,49]. Another interesting result could be neurofilament alterations observed immediately after PCPAgained by taking into account the different sources of 5HT treatment, during S-100b intracellular accumulation, couldinnervation: hippocampus receives 5HT innervation from be due to the reduction of extracellular S-100b. Themedian raphe predominantly, cortex receives innervation complete idea is summarized in Fig. 12.from dorsal and median raphe [9,50]. Both cortex and Obviously our speculations should be verified by ahippocampus have recovered the control levels of S-100b biochemical quantification of extracellular S-100b, duringimmunostaining in the T2 group showing a very similar these events. We believe that our in vivo model with aprofile. On the other hand striatum, predominantly inner- morphometric semiquantitative approach allows a finevated by fibers from the DRN [9,50], does not recover anatomical resolution. We are currently performing someS-100b control levels in the T2 group. experiments to confirm the S-100b release by studying in

The neuronal cytoskeleton presents a triplet of neurofila- vivo other biological effects described in vitro [21,22]. Thement proteins that are the main determinants of axonal presented 5HT-depletion model that implies a S-100b

caliber [19]. Altered neurofilament expression may lead to intracellular accumulation, with a consequent reduction inneurodegeneration [23]. We analyzed the expression of two the availability of the growth factor can be interesting toneurofilaments: Nf-200 which is present in mature axons study in vivo biological effects of S-100b. It would beand Nf-68 which increases during neurite outgrowth and useful to complement findings already described in vivo inmaturation. After PCPA treatment we found reduced Nf- transgenic mouse [53] and in vitro [21,22]. The S-100b

200 immunostaining in the T0 (cortex, hippocampus) or in biological effects are important and of potential clinicalthe T1 group (striatum). Morphological alterations of Nf- relevance, considering that over expression of S-100b has200 were observed especially in the striatal patches, also been related with Down’s syndrome and Alzheimer’scalled striosomes. Abnormal neurofilaments present in- disease [14,15,53] which are characterized by glial re-

12 A.J. Ramos et al. / Brain Research 883 (2000) 1 –14

Fig. 12. Proposed sequence of events in the recovering after PCPA-induced 5HT depletion. Normal: in the normal brain tryptophan (Trp) is hydroxylatedby the TrpOH (the rate limiting enzyme in the 5HT synthesis) and decarboxylated by the aromatic amino acid decarboxylase (AADC) to give 5HT.Released 5HT interacts with 5HT receptor in glial cells. By an unknown mechanism S-100b is released from glial cells. Extracellular S-100b stabilizes1A

cytoskeleton and maintains innervation. Depleted: during 5HT depletion the activity of TrpOH is reduced, giving a lower 5HT release. There is loweractivation of 5HT receptors and minor S-100b release, S-100b is accumulated inside glial cells giving increased S-100b immunostaining. The low1A

availability of extracellular S-100b produces alterations in the neuronal cytoskeleton and the reduction of Nf-68, Nf-200 and 5HT-T positive fibers.Recovering: an important pool of 5HT is synthesized because of the increased TrpOH expression [41]. 5HT produces activation of 5HT receptors and the1A

S-100b release. A reduction of intracellular S-100b immunostaining is observed. The effect of the S-100b release could be the normalization ofcytoskeleton and the sprouting of fibers.

giant: anatomy and plasticity of the brain serotonergic system, J.action, S-100b accumulation and neuronal morphologicalClin. Psychiatry 52 (Suppl.) (1991) 4–16.and functional alterations.

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