Laboratory of Neuroscience, Department of Physiology and , 1 Service of Neurology, Libera Università Campus Bio-Medico and , 2 Department of Psychology, Università La Sapienza, Rome, Italy
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The rodent somatosensory system, with its one-to-one correspondence between each vibrissa and its cortical barrel-like projection area, represents an ideal model to assess the impact of serotoninergic manipulations on brain development and plasticity [for review see (Rice, 1995; Killackey et al., 1995
)]. Furthermore, transient barrel-like distribution of 5-HT (Fujimiya et al., 1986
; D'Amato et al., 1987
; Rhoades et al., 1990
; Blue et al., 1991
; Bennett-Clarke et al., 1991
, 1994a
; Dori et al., 1996
), of 5-HT1B and 5-HT2A receptors (Leslie et al., 1992
; Bennett-Clarke et al., 1993
; Mansour-Robaey et al., 1998
) and of the 5-HT transporter (D'Amato et al., 1987
; Lebrand et al., 1996
; Mansour-Robaey et al., 1998
) [for review see (Fuchs, 1995
)] in layer IV of neonatal rodent somatosensory cortex (SSC) spur further interest into 5-HT involvement in the development of thalamocortical pathways.
Initial support for neurotrophic roles of 5-HT in mammalian somatosensory pathways has come from pharmacologically induced 5-HT depletion (Blue et al., 1991; Bennett-Clarke et al., 1994b
; Osterheld-Haas et al., 1994
). Systemic administration of a variety of 5-HT-depleting agents has been shown to produce alterations of barrel-like patterns, best described as a delay in barrel pattern maturation (Blue et al., 1991
; Osterheld-Haas et al., 1994
) or as a reduction in cross-sectional areas of whisker barrels (Bennett-Clarke et al., 1994b
).
Recent support for the involvement of 5-HT in thalamocortical development has come from transgenic models. MAO-A knock- out mice are devoid of cortical barrels, which reappear when 5-HT synthesis is inhibited by p-chlorophenylalanine (PCPA) (Cases et al., 1996). Furthermore, mice devoid of the plasma membrane 5-HT transporter (Bengel et al., 1998
) display significantly thinner barrels in layer IV of the postero-medial barrel subfield (PMBSF) and the absence of barrel-like patterns in the other subfields of the primary somatosensory cortex (Persico et al., 1998
).
Further interest in these data, underscoring 5-HT roles in cortical neurodevelopment, is spurred by developmental changes in brain 5-HT content and 5-HT synthesis capacity demonstrated in rhesus monkeys (Goldman-Rakic and Brown, 1982) and in humans (Chugani et al., 1999
). Moreover, these results are in line with in vivo evidence of transiently increased neonatal 5-HT levels in other brain regions of non-rodent species, possibly related with 5-HT regulation of synaptogenesis (Okado et al., 1989
, 1993
; Chen et al., 1994
; Niitsu et al., 1995
) and with previous in vitro studies showing 5-HT effects on cortical synaptogenesis, neurite branching, myelination and glial proliferation in tissue culture (Chubakov et al., 1986
; Sikich et al., 1990
). More recent in vitro studies focused on thalamocortical neurons suggest that 5-HT may influence both neurite outgrowth and synaptic transmission in neonatal rats (Rhoades et al., 1994
; Lieske et al., 1999
). Interestingly, 5,7-DHT-induced barrel pattern alterations in vivo appear to be tetrodoxin (TTX)- insensitive (Rhoades et al., 1998
).
Although both 5-HT depletion studies and assessments of brains from knockout mice provide converging support for 5-HT roles in somatosensory thalamocortical neurodevelopment, caution is raised by systemic 5-HT-depleting drug treatments inevitably inducing some degree of growth impairment, possibly through malnutrition, which has been shown to delay whisker barrel pattern formation per se (Vongdokmai, 1980). Our study was undertaken to provide an estimate of the impact of (i) 5-HT depletion and (ii) delayed body growth on development of the whisker barrel cortex, following systemic 5-HT-depleting drug treatments. To this aim, we systemically administered at birth either the 5-HT terminal-selective neurotoxin p-chloroamphetamine (PCA) (Miller et al., 1970
; Baumgarten et al., 1982
; Commins et al., 1987
; Haring et al., 1994
) [for review see (Azmitia and Whitaker-Azmitia, 1995
)] or the 5-HT synthesis inhibitor p-chlorophenylalanine (PCPA) (Koe and Weissman, 1966
), whose effects on cortical 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) levels, on whisker barrel cross-sectional areas, and on body and brain weight were assessed in the same animal for the first time in this study. Furthermore, in order to better define protein deficiency contributions to drug-induced delays in barrel formation, we assessed with the same methodology the off-spring of pregnant females fed with either a hypoproteic or a normoproteic diet starting just prior to the expected birth date and throughout the first week of neonatal life of their pups. Our results clearly show that when pharmacological interventions yield both 5-HT depletion and neonatal growth retardation, the latter factor is largely responsible for altered somatosensory cortical development. Moreover, drug or dietary treatments that do not reduce brain weight also do not have an impact on barrel size, despite yielding variable extents of cortical 5-HT depletion.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Experiments were performed on newborn Lewis rats (Charles River, Calco, Italy) during the first 15 days of postnatal life. In this study, P0 is defined as the first 24 h after delivery. Pregnant female rats were singly housed and maintained on a 12 h light/12 h dark schedule (lights on at 7:00 a.m.), with free access to food and water.
Systemic Administrations of PCA and PCPA
All animals were injected between 3 and 6 h after birth. PCA experiments were carried out on 17 pups belonging to two litters; PCPA experiments were performed on a separate set of 47 animals belonging to four litters. Each litter was divided into an experimental and a control group. Pups were weighted and briefly anesthetized using hypothermia. Experimental animals from the first two litters were injected s.c. with either 3.5 or 14 mg/kg PCA (0.01 ml/g of a 0.35 or 1.4 mg/ml solution in saline, respectively); experimental animals from the remaining four litters received PCPA (300 mg/kg s.c.; 0.01 ml/g of a 30 mg/ml solution). Control animals were injected with equivalent volumes of sterile saline solution. After the injection, pups were quickly warmed using an infrared light and returned to their cage.
Manipulation of Dietary Protein Intake
Since PCA and PCPA administration produced prominent decreases in body and brain growth (see Results), possible contributions of early- postnatal hypoproteic dietary intake to altered barrel development were assessed in 12 pups, belonging to two separate litters. The diets administered to pregnant females were identical to those adopted by Vongdokmai (Vongdokmai, 1980) in mice. Two female Lewis rats undergoing timed pregnancies were fed with a normoproteic 28%-casein diet throughout their pregnancy. Two days prior to the expected delivery date, one of the two females was switched to a hypoproteic 8%-casein diet, including increased amounts of carbohydrates to maintain constant the total caloric intake. Both animals were fed ad libitum with either the normoproteic or the hypoproteic diet, until their pups reached P6.
Assessments of Brain Weights and Cortical Dissection
Animals were killed by decapitation either at P6 (PCA- or PCPA-treated vs. saline; hypoproteic vs. normoproteic diet) or at P14 (PCPA-treated vs. saline). Brains were quickly dissected and the hindbrain removed with a coronal cut separating dorsally the superior and inferior colliculi, and reaching ventrally the mamillary bodies (Glowinski and Iversen, 1966). Brains were immediately weighted, the hemispheres separated with a scalpel and the cortices dissected from the rest of each hemisphere. Whisker barrel fields were punched out from left hemispheres using a template (Strominger and Woolsey, 1987
), immediately frozen on dry ice (<4 min after sacrifice), stored at 70°C for a maximum of 2 weeks and then placed in liquid nitrogen until later assessments using HPLC. The whole controlateral cortex was quickly placed free-floating in 4% paraformaldehyde dissolved in phosphate buffer (PB, pH 7.4) at 4°C for 1216 h, flattened, cryoprotected with 30% sucrose dissolved in PB (pH 7.4) for 3648 h, cut into 50 µm slices using a cryostat and processed for acethylcholinesterase (AchE) staining (see below).
Assessment of Neurotransmitter Levels in the SSC
5-HT, 5-HIAA and norepinephrine (NA) were simultaneously determined utilizing a reverse-phase HPLC procedure coupled with electrochemical detection (Kempf and Mandel, 1981; Cabib and Puglisi-Allegra, 1994
). On the day of analysis, frozen samples were weighted and homogenized in 0.1 N HClO4 containing 6 mM Na-metabisulphite and 1 mM EDTA. To compensate for variations in sample size, the extraction solution was added to each sample at a concentration of 10 µl/mg of sample weight. Homogenates were centrifuged at 10 000 g for 20 min at 4°C. Supernatants were removed and stored in the dark on ice until a 15 µl aliquot of each sample was transferred to the HPLC system, consisting of a Waters 460 electrochemical detector with a glass carbon working electrode and a pump (Waters 510). The potential was set at 800 mV (vs. AgAgCl reference electrode). A Bondapak C18 column (10 mm particle size, 300 x 3.1 mm i.d.) purchased from Waters Assoc. (Millipore, Milford, MA) was employed. The flow rate was 1.1 ml/min. The mobile phase consisted of 8% methanol in 0.1 M Na-phosphate buffer (pH 3.0), 0.01 mM Na2EDTA and 1.1 mM L-octane sulphonic acid sodium salt (Aldrich, Milwaukee, WI); 3,4-dihydroxy-benzylamine hydrobromide (Aldrich) was used as internal standard.
Labelling Thalamocortical Terminals by AchE Histochemistry
AchE has been shown to transiently label somatosensory thalamocortical afferents in neonatal rodents [for review see (Fuchs, 1995)]. Histochemistry for AchE was performed according to the method of Hedreen et al. (Hedreen et al., 1985
), achieving on these post-fixed brains results qualitatively similar to those routinely obtained from brains of perfused animals (Fig. 1
). Briefly, slices were rinsed in 0.1 M sodium acetate (pH 6.0) and incubated for 5 h at 37°C in a medium containing 50 mg of acetylthiocholine iodide, 65 ml of 0.1 M sodium acetate (pH 6.0), 4 ml of 0.1 M sodium citrate, 10 ml of 0.03 M cupric sulfate (CuSO45H2O), 21 ml of distilled water and 100 µl of a 102 M solution of tetraisopropylpyrophosphoramide (final concentration 105 M) for 100 ml of total medium volume. Slices were then rinsed with agitation for 10 min in 0.1 M sodium acetate (pH 6.0), incubated for 10 min in 2% potassium ferrocyanide at room temperature and rinsed twice in 0.1 M sodium acetate (pH 6.0).
|
AchE-stained sections were used to measure single barrel cross-sectional areas and total PMBSF surface using the Kontron Imaging System KS100 (Kontron Elektronik, Eching b. München, Germany). Following image acquisition, contrast and smoothing were adjusted to yield as well- defined and uniform barrel boundaries as possible in each preparation. All whisker barrels within the PMBSF of the primary somatosensory cortex were circled and areas were measured by one of the authors, who was blind to the treatment group. The following whisker barrel areas were used for statistical analyses: A1A4, B1B4, C1C7, D1D8, E1E8. Data from barrels , ß,
,
, A5 and higher-order barrels from rows D and E were not included in the analyses, due to interindividual inconsistencies in barrel presence or to frequent unreliability of barrel boundaries. The cortical surface devoted to the PMBSF was measured as (i) PMBSF area expressed in mm2, encompassing all of the barrels listed above together with barrels
, ß,
,
; and (ii) total line length expressed in mm, obtained connecting the barrel centroids of the four most caudal vibrissae in each row, as described previously (Bennett-Clarke et al., 1994b
). PMBSF area thus provides a direct measure of cortical surface devoted to AchE-stained vibrissa-related patterns, whereas total line length yields a relative estimate of PMBSF area, controlling for differences in the extent of arborization present in the outermost barrels, as first suggested by Riddle et al. (Riddle et al., 1992
, 1995
).
Statistical Analyses
A one-way analysis of variance (ANOVA) was performed to assess systemic PCA-induced alterations in each neurochemical, histological and physical parameter. When significant treatment effects were detected, pairwise a priori contrasts were performed, testing the hypothesis that PCA would reduce cortical 5-HT content, barrel areas and body growth, as expected on the basis of previous work (Blue et al., 1991; Osterheld-Haas et al., 1994
). Independent t-tests were used to compare systemic PCPA vs. saline and hypoproteic vs. normoproteic diet effects on each neurochemical, histological and physical parameter.
Correlation coefficients between single variables and stepwise multiple regressions were finally performed to estimate how much of the variance in mean whisker barrel area is explained by differences in brain weight and how much stems from cortical 5-HT content (Tabachnick and Fidell, 1989). Entry and removal of independent variables into the regression equation were defined setting F-to-enter = 1 and F-to-remove = 0, respectively. The percentage variance in mean whisker barrel area explained by changes in brain weight and/or by extent of 5-HT depletion was derived from the change in R2 brought about by the entry of each of the two independent variables into the regression equation.
Statistical significance was set at < 0.05 also for a priori contrasts. Sample sizes were established as much as possible prior to experiments, using power analyses performed with the POWER program (Dupont and Plummer, 1990
). Parameters were set at
< 0.05, and ß > 0.80, while interindividual variability was estimated from available literature or original preliminary data. Results are reported as mean ± SEM.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Single injections of PCA (3.5 or 14 mg/kg s.c.) performed at P0 yielded statistically significant 49.0 and 58.9% reductions in somatosensory cortical 5-HT content, respectively, at P6 (Fig. 2). In these same animals, 5-HIAA tissue levels were decreased by 24.440.0%, yielding significantly increased 5-HIAA/5-HT ratios (Fig. 2
). The 14 mg/kg dose did not yield changes in 5-HT content, 5-HIAA content and 5-HIAA/5-HT ratio significantly more profound than those produced by the 3.5 mg/kg dose. No significant effect on NA tissue content was recorded.
|
Statistically significant differences in body weight between PCA- and saline-injected animals were recorded at P6 but not at P0, due to different growth rates (Fig. 2). Compared with saline -injected controls, PCA-treated animals displayed significantly reduced mean body weight [25.732.1% for lower and higher dose, respectively; one-way ANOVA: F(2,16) = 8.30, P < 0.001] and brain weight [9.6% decrease for the higher dose; one-way ANOVA: F(2,16) = 5.89, P < 0.05] before sacrifice at P6.
Effects of Neonatal Systemic PCA Administration on Whisker Barrel Areas
Single PCA injections at P0 yielded significant (9.021.4%) reductions in mean whisker barrel areas, with the higher dose significantly more effective than the lower dose [one-way ANOVA: F(2,16) = 18.92, P < 0.001; a priori contrast (3.5 vs. 14 mg/kg): T(14) = 6.15, P < 0.001; Fig. 2]. Assessments of single whisker barrel rows indicate that all barrel rows were affected to the same extent (data not shown). Significant reductions in cortical surface devoted to the AchE-stained vibrissae-related pattern were also recorded, measured either as PMBSF area [one-way ANOVA: F(2,16) = 12.58, P < 0.001] or total line length [one-way ANOVA: F(2,16) = 7.47, P < 0.01; Fig. 2
]. The correlation between PMBSF area and total line length was significant (Pearson r = 0.84, P < 0.001).
Effects of Neonatal Systemic PCPA Administration on Monoamine Levels in the SSC
Since PCA may also exert direct toxic effects on thalamocortical terminals, which transiently express the 5-HT transporter (Lebrand et al., 1996) (see Discussion), the next series of experiments was undertaken to indirectly estimate the weight of reduced growth rates and 5-HT depletion on barrel formation, while eliminating the potential for direct thalamocortical neurotoxicity.
Single injections of the selective tryptophan-hydroxylase inhibitor PCPA (300 mg/kg s.c.) (Koe and Weissman, 1966), performed during the first 6 h after delivery, produced massive mean decreases in somatosensory cortical 5-HT and 5-HIAA levels (93.4 and 83.3%, respectively) measured at P6 (Fig. 3
). As with PCA, systemic PCPA was more effective in reducing 5-HT than 5-HIAA levels, yielding 5-HIAA/5-HT ratios increased by ~150% (Fig. 3
).
|
|
PCPA again produced relevant effects on body and brain weight, assessed at P6 (Fig. 3). PCPA-injected animals displayed even more prominent (37.6%) decreases in body weight, with striking (71.2%) reductions in body growth rate compared with saline- injected controls. Reductions in body weight and growth rates recorded at P6 were accompanied by significant (22.5%) reductions in brain weight as well (Fig. 3
).
PCPA effects on body growth were less pronounced though still significant at P14, with 21.1% reductions in body weight and growth rates decreased by 30.8% (Fig. 4). Brain weights were practically identical, with only a 3.6% mean decrease in PCPA-treated animals compared to saline-injected pups (Fig. 4
).
Effects of Neonatal Systemic PCPA Administration on Whisker Barrel Areas
Systemic PCPA administration at P0 produced highly significant (19.4%) reductions in mean whisker barrel areas at P6, coupled with 17% and 9.7% reductions in PMBSF area and total line length, respectively (Fig. 3). Decreases of similar extent were recorded in all barrel rows, ranging from ~16% for barrels in rows A and B to 21% for rows D and E.
At P14, modest (6.6%) reductions in mean barrel size of PCPA-injected pups were not statistical significant (Fig. 4). Also, non-significant 7.2% decreases in PMBSF area were associated with measures of total line length practically superimposable on those of saline-treated animals (Fig. 4
).
Correlations between PCPA Effects on Cortical 5-HT Levels, Brain Weight and Whisker Barrel Areas
Since PCPA does not possess a potential for direct neurotoxic effects, it was possible to estimate the impact of the two independent variables, namely brain weight and 5-HT content in the SSC, on the dependent variable, mean whisker barrel area, using correlation and stepwise multiple regression analyses. Results of these analyses are summarized in Table 1. Mean whisker barrel area values of animals for the entire P0P6 sample, including 13 PCPA- and 15 saline-injected pups, are significantly correlated with brain weight (Pearson r = 0.82, P < 0.001), but not with cortical 5-HT content (r = 0.21, n.s.). Saline-treated animals show expected positive correlations between mean whisker area and brain weight (Pearson r = 0.60, P < 0.05), but not with cortical 5-HT content (r = 0.36, n.s.). These data derive from two distinct replicas of the P0P6 experiment performed on two separate litters (litter A: n = 16, nine PCPA and seven saline; litter B: n = 12, six PCPA and six saline), whose results are displayed in Figure 5
. PCPA-treated animals from both litters clearly show a positive linear correlation between mean whisker barrel area and brain weight values, and no correlation between barrel areas and cortical 5-HT content (Fig. 5
). No significant correlation is consistently found in saline-treated animals from both litters sacrificed at P6 (Fig. 5
).
|
|
Stepwise multiple regression analyses performed on the entire P0P6 sample of 28 animals indicate that brain weight explains 84.4% of the variance and that somatosensory cortical 5-HT content does not reach the threshold to fit into the model, as it would explain only 05% of the variance (Table 1). Both litters A and B, analyzed separately, display identical trends (Fig. 5
). Analyses performed on the P0P14 sample yield a very similar outcome, with brain weight explaining 51.6% of the variance and somatosensory cortical 5-HT content again not reaching the threshold to fit into the regression equation (Table 1
).
Effects of Decreased Protein Intake on Neonatal Monoamine Levels in the SSC, Body and Brain Weight, and on Whisker Barrel Areas
Results summarized in the previous sections indicate that PCA and PCPA effects on whisker barrel size may largely stem from drug-induced reductions in body and brain growth rates. In order to assess potential contributions of malnutrition and diminished protein intake to these effects, we appraised the alterations produced by a hypoproteic diet fed to a pregnant female starting 2 days prior to the expected delivery date and throughout the first week of postnatal life of her offspring. This protocol was chosen to ensure that protein intake would be reduced starting at approximately the same developmental stage affected by PCPA inections, i.e. at P0.
Compared with seven pups of a female rat fed the normo-proteic-normocaloric diet, five pups of a female fed the hypoproteic-normocaloric diet showed highly significant (41.8 and 47.6%) decreases in somatosensory cortical 5-HT and 5-HIAA levels, respectively (Fig. 6). Also, somatosensory cortical NA levels were reduced by 19.33%. Despite significantly lower cortical 5-HT levels and significant (17.7%) reductions in body weight at P6, negligible (2.6%) decreases in brain weight were interestingly paralleled by similarly negligible (2.9%) reductions in mean barrel area (Figs 6 and 7
).
|
|
Mean whisker barrel area values of animals from the entire sample, including five malnourished and seven control pups, were significantly correlated with brain weight (Pearson r = 0.78, P < 0.01), but not with cortical 5-HT content (r = 0.06, n.s.), as summarized in Table 2. Both malnourished and control pups, analyzed separately, yielded significant correlations between mean whisker barrel areas and brain weight, but no correlation with cortical 5-HT content (Table 2
).
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The results reported here were achieved by concomitantly assessing body and brain weights, monoamine levels in the SSC and whisker barrel areas in the same animal following systemic drug administration or manipulation of dietary protein intake. Parallel measurements of neurotransmitter levels in the SSC of one hemisphere and of whisker barrel size in the controlateral hemisphere greatly enhance the reliability of our results and their statistical power.
The impact of inter-individual and particularly inter-litter variability was significantly reduced by using inbred Lewis rats instead of outbred strains. Genetic homogeneity has further strengthened the reliability of our findings, by contributing to the minimal variability in monoamine and metabolite levels, barrel and PMBSF size, and physical parameters encountered in this study (see Figs 24 and 6).
Systemic PCA experiments employing two distinct doses were performed to verify the coincidence of 5-HT depletion and weight loss using several different 5-HT-depleting drug paradigms; however, only results obtained with PCPA, a selective tryptophan-hydroxylase inhibitor, were used for multiple regression analyses (Table 1 and Fig. 5
). Systemic PCA data were excluded from these analyses because of the potential confounding factor of PCA-mediated direct damage of thalamocortical endings, which transiently express both the plasma membrane 5-HT transporter and the vesicular monoamine transporter in neonatal rodents (Lebrand et al., 1996
). PCA can be expected to cause massive 5-HT release from terminals of mostly dorsal raphe neurons providing diffuse innervation of the somatosensory cortex (Bennett-Clarke et al., 1991
; Lebrand et al., 1996
). Favored by PCA-induced MAO inhibition, extracellular 5-HT is believed to be non-enzymatically converted into 5,6-DHT, a neurotoxic compound selectively taken up by 5-HT transporter- expressing terminals; once inside the terminal, 5,6-DHT rapidly autoxidizes forming highly reactive quinones, which spontaneously cross-link intracellular proteins, causing neurite degeneration (Miller et al., 1970
; Baumgarten et al., 1982
; Commins et al., 1987
; Haring et al., 1994
) [for review see (Azmitia and Whitaker-Azmitia, 1995
)]. The transient expression of the 5-HT transporter and of the vesicular monoamine transporter during the first 2 weeks of neonatal life in rodents could thus expose thalamocortical terminals to neurotoxic damage by drugs, such as PCA, active through uptake-dependent mechanisms. Preliminary results using PCA-delivering elvax chips placed directly over the SSC of neonatal rats indeed provide initial support to this hypothesis (Persico et al., 1997
) (unpublished observations).
One practical limitation inherent to our experimental approach is that only one staining can be used to label thalamocortical terminals, when working with manageable sample sizes. Histochemistry for AchE was employed here for its rapidity, reliability and validity. Our histochemical procedure was adjusted to consistently yield, in unperfused tissues fixed only by immersion, results comparable to those obtained using perfused brains (Fig. 1). Furthermore, AchE has been shown to be a valid and reliable marker of thalamocortical terminals in neonatal rats: it is transiently expressed by thalamocortical neurons between P3 and P18, and somatosensory cortical AchE corresponds precisely to areal and laminar distributions of thalamocortical terminals (Kristt, 1979
; Kristt and Waldman, 1981
; Robertson, 1987
). Nonetheless, DiI or tenascin staining should be employed in future experiments similar to those described here, to further strengthen our conclusions.
In malnutrition experiments, dietary protein intake may have been higher than anticipated solely on the basis of the hypoproteic-normocaloric 8%-casein diet, as cannibalism of dead pups by their mother occurs frequently in rodents during the first few days after delivery. Nonetheless, the significant decreases in somatosensory cortical 5-HT and NA levels recorded in malnourished pups (Fig. 6) strongly suggest that the diet effectively reduced amino acid intake and consequently brain monoamine synthesis.
Finally, most studies performed to date, including ours, focus on cross-sectional barrel areas, which may not represent the most valid and/or sensitive parameter to assess 5-HT modulation of thalamocortical development in the somatosensory system. Direct assessments of neurite arborization patterns, for example, may yield more interpretable results.
Effects of 5-HT Depletion on Neonatal Growth: Comparison between Our Findings and Previous Literature
The involvement of 5-HT in development of the mammalian neocortex is supported by evidence coming from both in vitro and in vivo studies. Our results confirm prior findings (Blue et al., 1991; Osterheld-Haas et al., 1994
; Bennett-Clarke et al., 1994b
) indicating that systemic 5-HT depleting treatments produce an overall maturational delay, as supported here by brain weights and cross-sectional areas of whisker barrels being significantly reduced at P6, but not at P14 (Figs 24
). This study, however, points toward an interpretation of in vivo results neither simple nor straightforward, and spurs interest in a reappraisal of in vivo studies employing 5-HT depleting drugs, both in neonatal rodents (Blue et al., 1991
; Bennett-Clarke et al., 1994b
; Chen et al., 1994
; Osterheld-Haas et al., 1994
) and in other species (Okado et al., 1993
; Niitsu et al., 1995
).
This study reaches three conclusions: firstly, the body and brain weights of PCA- or PCPA-injected neonatal rats increase between P0 and P6 significantly less than those of saline-treated pups; secondly, when 5-HT depletion and decreased brain growth are present, delayed maturation of thalamocortical pathways induced by impaired brain growth is clearly dominant over selective 5-HT-mediated modulation of thalamocortical pathways (Fig. 5 and Table 1
); and thirdly, drug or dietary treatments that produce significant reductions in cortical 5-HT levels without prominently affecting brain weight (i.e. PCPA-injected animals at P14 and neonatal malnutrition) yield minimal or no impact on whisker barrel size (Figs 4, 6 and 7
and Table 2
).
The effect of 5-HT-depleting drugs on body and brain weights is believed to stem mostly from decreased food intake, as suggested by 5-HT1B and 5-HT2C receptor-mediated modulation of feeding behaviors, affecting both hypothalamic and extrahypothalamic sites (Lucas et al., 1998). Nonetheless, contributions from altered metabolic rates are supported by the relatively modest impact of the hypoproteic diet on body weight compared with the effect of PCA- and PCPA-treatment.
Studies assessing 5-HT impact on development of thalamocortical pathways using 5-HT-depleting agents such as PCA or 5,7-DHT usually report modest-to-moderate decreases in body weight (Blue et al., 1991; Osterheld-Haas et al., 1994
) in the absence of significant decreases in brain weight (Blue et al., 1991
; Bennett-Clarke et al., 1994b
; Osterheld-Haas et al., 1994
). The brain is well known to be one of the organs that least suffers from malnutrition merely in terms of decreased organ weight, possibly due to brain growth sparing mechanisms involving enhanced amino acid transport through the bloodbrain barrier (Desai et al., 1996
). Despite these compensatory mechanisms, malnutrition has been shown to trigger significant alterations in brain metabolism, involving, for example, fatty acids (Marin et al., 1995
), 5-HT synthesis and release (Chen et al., 1992
; Blatt et al., 1994
; Manjarrez et al., 1994
, 1996
), as well as noradrenergic turnover (Soto-Moyano et al., 1995
), which do not necessarily reverse following nutritional recovery (Manjarrez et al., 1994
, 1996
). Furthermore, the time course of drug-induced maturational delays reported in some studies (Blue et al., 1991
; Osterheld-Haas et al., 1994
) is superimposable on the time course of delayed thalamocortical development induced by malnutrition (Vongdokmai, 1980
). Finally, these studies also pose the problem of potential direct damage to thalamocortical terminals, which cannot be immediately distinguished from modulation of terminal arborization and barrel formation by 5-HT. Direct damage, for example, could readily explain persistent changes in whisker barrel areas that are maintained into adulthood (Bennett-Clarke et al., 1994b
), as well as TTX- insensitivity of 5,7-DHT-induced reductions in mean barrel areas (Rhoades et al., 1998
).
PCPA doses administered in studies assessing 5-HT roles in synaptogenesis were usually higher than the dose employed here and/or administered repeatedly over several days (Okado et al., 1993; Chen et al., 1994
; Niitsu et al., 1995
). Treatment protocols of this sort may also yield prominent reductions in body and brain growth, although inter-species differences in sensitivity to drug effects on weight may be present. Our results suggest caution in attributing reductions in synaptic density entirely to decreased 5-HT content, as weight loss may have contributed to this effect or may even possibly account for it, following the most aggressive treatments.
All pharmacological treatments employed here may have produced decreases in cortical 5-HT content below the threshold necessary for serotoninergic modulation of thalamocortical development. However, prior studies have employed either doses higher than the ones administered here (Blue et al., 1991; Osterheld-Haas et al., 1994
) or selected animals displaying more profound 5-HT depletions than those recorded in our PCA-treated sample (Bennett-Clarke et al., 1994b
). Some of our PCPA-treated animals clearly display very profound 5-HT depletions in the presence of barrel areas within the range of saline-injected pups (Fig. 5
).
In conclusion, postnatal brain growth appears to be a major determinant of barrel field development, while postnatal reduction in 5-HT brain levels following pharmacological or dietary interventions in vivo appear to exert rather minor direct effects on barrel development, if any. This outcome is remarkably different from the major alterations in somatosensory cortical cytoarchitecture produced by early postnatal exposure to 5-HT excess both in MAO-A and 5-HT transporter knock-out mice (Cases et al., 1996; Lebrand et al., 1996
; Persico et al., 1998
). Furthermore, the difficulty in disentangling 5-HT neurodevelopmental roles from the consequences of drug-induced growth impairment and from direct neurotoxic damage of thalamocortical terminals may further support the preferential use of transgenic mouse models (Cases et al., 1996
; Lebrand et al., 1996
; Persico et al., 1998
) over pharmacologic 5-HT depletion paradigms as a means to reliably assess 5-HT impact on neocortical development. Interestingly, experiments using 5-HT transporter knock-out animals indicate that the critical period for 5-HT modulation of thalamocortical terminal growth into the somatosensory cortex may be largely limited to the first 48 h after birth in mice (A.M. Persico, F. Keller, K.P. Lesch and D. Murphy, manuscript in preparation), as found for systemic PCA- and PCPA-injected animals (A.M. Persico and F. Keller, unpublished observation). However, in knock-out mice 5-HT excess does appear to modulate thalamocortical pattern formation in the absence of effects on body and brain growth, and of changes in amount of cortical surface devoted to the PMBSF (Persico et al., 1998
).
![]() |
Notes |
---|
Address correspondence to Flavio Keller, MD, Laboratory of Neuroscience, L.U.C.B.M., Via Longoni 83, I-00155 Rome, Italy. Email f.keller{at}unicampus.it.
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bailey CH, Chen MC, Keller F, Kandel ER (1992) Serotonin-mediated endocytosis of apCAM: an early step of learning-related synaptic growth in Aplysia. Science 256:645649.[ISI][Medline]
Baumgarten HG, Klemm HP, Sievers J, Schlossberger HG (1982) Dihydroxytryptamines as tools to study the neurobiology of serotonin. Brain Res Bull 9:131150.[ISI][Medline]
Bengel D, Murphy DL, Andrews AM, Wichems CH, Feltner D, Heils A, Moessner R, Westphal H, Lesch KP (1998) Altered brain serotonin homeostasis and locomotor insensitivity to 3,4-methylenedioxymethamphetamine (Ecstasy) in serotonin transporter-deficient mice. Mol Pharmacol 53:649655.
Bennett-Clarke CA, Chiaia NL, Crissman RS, Rhoades RW (1991) The source of the transient serotoninergic input to the developing visual and somatosensory cortices in rat. Neuroscience 43:163183.[ISI][Medline]
Bennett-Clarke CA, Leslie MJ, Chiaia NL, Rhoades RW (1993) Serotonin 1B receptors in the developing somatosensory and visual cortices are located on thalamocortical axons. Proc Natl Acad Sci USA 90:153157.[Abstract]
Bennett-Clarke CA, Hankin MH, Leslie MJ, Chiaia NL, Rhoades RW (1994a) Patterning of the neocortical projections from the raphe nuclei in perinatal rats: investigation of potential organizational mechanisms. J Comp Neurol 348:277290.[ISI][Medline]
Bennett-Clarke CA, Leslie MJ, Lane RD, Rhoades RW (1994b) Effect of serotonin depletion on vibrissa-related patterns of thalamic afferents in the rat's somatosensory cortex. J Neurosci 14:75947607.[Abstract]
Blatt GJ, Chen JC, Rosene DL, Volicer L, Galler JR (1994) Prenatal protein malnutrition effects on the serotoninergic system in the hippocampal formation: an immunocytochemical, ligand binding, and neuro- chemical study. Brain Res Bull 34:507518.[ISI][Medline]
Blue ME, Erzurumlu RS, Jhaveri S (1991) A comparison of pattern formation by thalamocortical and serotoninergic afferents in the rat barrel field cortex. Cereb Cortex 1:380389.[Abstract]
Cabib S, Puglisi-Allegra S (1994) Opposite responses of mesolimbic dopamine system to controllable and uncontrollable aversive experiences. J Neurosci 14:33333340.[Abstract]
Cases O, Vitalis T, Seif I, De Maeyer E, Sotelo C, Gaspar P (1996) Lack of barrels in the somatosensory cortex of monoamine oxidase A-deficient mice: role of a serotonin excess during the critical period. Neuron 16:297307.[ISI][Medline]
Chen JC, Tonkiss J, Galler JR, Volicer L (1992) Prenatal protein malnutrition in rats enhances serotonin release from hippocampus. J Nutr 122:21382143.[ISI][Medline]
Chen L, Hamaguchi K, Ogawa M, Hamada S, Okado N (1994) PCPA reduces both monoaminergic afferents and nonmonoaminergic synapses in the cerebral cortex. Neurosci Res 19:111115.[ISI][Medline]
Chubakov AR, Gromova EA, Konovalov GV, Sarkisova EF, Chumasov EI (1986) The effects of serotonin on the morpho-functional development of rat cerebral neocortex in tissue culture. Brain Res 369: 285297.[ISI][Medline]
Chugani DC, Muzik O, Behen M, Rothermel R, Janisse JJ, Lee J, Chugani HT (1999) Developmental changes in brain serotonin synthesis capacity in autistic and nonautistic children. Ann Neurol 45:287295.[ISI][Medline]
Commins DL, Axt KJ, Vosmer G, Seiden LS (1987) Endogenously produced 5,6-dihydroxytryptamine may mediate the neurotoxic effects of para-chloroamphetamine. Brain Res 419:253261.[ISI][Medline]
D'Amato RJ, Blue ME, Largent BL, Lynch DR, Ledbetter DJ, Molliver ME, Snyder SH (1987) Ontogeny of the serotoninergic projection to rat neocortex: transient expression of a dense innervation to primary sensory areas. Proc Natl Acad Sci USA 84:43224326.[Abstract]
Desai M, Crowther NJ, Lucas A, Hales CN (1996) Organ-selective growth in the offspring of protein-restricted mothers. Br J Nutr 76:591603.[ISI][Medline]
Dori I, Dinopoulos A, Blue ME, Parnavelas JG (1996) Regional differences in the ontogeny of the serotoninergic projection to the cerebral cortex. Exp Neurol 138:114.[ISI][Medline]
Dupont WD, Plummer WJ Jr (1990) Power and sample size a review and computer program. Controlled Clin Trials 11:116128.[ISI][Medline]
Fuchs JL (1995) Neurotransmitter receptors in developing barrel cortex. In: Cerebral cortex, Vol. 11. The barrel cortex of rodents (Jones EG, Diamond IT, eds), pp. 375409. New York: Plenum Press.
Fujimiya M, Kimura H, Maeda T (1986) Postnatal development of serotonin nerve fibers in the somatosensory cortex of mice studied by immunohistochemistry. J Comp Neurol 246:191201.[ISI][Medline]
Glanzman DL, Kandel ER, Schacher S (1990) Target-dependent structural changes accompanying long-term synaptic facilitation in Aplysia neurons. Science 249:799802.[ISI][Medline]
Glowinski J, Iversen LL (1966) Regional studies of catecholamines in rat brain. 1. The disposition of 3H-norepinephrine, 3H-dopamine and 3H-DOPA at various regions of the brain. J Neurochem 13:665669.
Goldman-Rakic PS, Brown RM (1982) Postnatal development of monoamine content and synthesis in the cerebral cortex of rhesus monkeys. Dev Brain Res 4:339349.[ISI]
Haring JH, Faber KM, Wilson CC (1994) Transient reduction in hippocampal serotoninergic innervation after neonatal parachloroamphetamine treatment. Dev Brain Res 83:142145.[ISI][Medline]
Hedreen JC, Bacon SJ, Price DL (1985) A modified histochemical technique to visualize acetylcholinesterase-containing axons. J Histo-chem Cytochem 33:134140.[Abstract]
Kempf E, Mandel P (1981) Reverse-phase high-performance liquid chromatographic separation and electrochemical detection of norepinephrine, dopamine, serotonin and related major metabolites. Anal Biochem 112:223231.[ISI][Medline]
Killackey HP, Rhoades RW, Bennett-Clarke CA (1995) The formation of a cortical somatotopic map. Trends Neurosci 18:402407.[ISI][Medline]
Koe BK, Weissman A (1966) p-Chlorophenylalanine: a specific depletor of brain serotonin. J Pharmacol Exp Ther 154:499516.[ISI][Medline]
Kristt DA (1979) Somatosensory cortex: acetylcholinesterase staining of barrel neuropil in the rat. Neurosci Lett 12:177182.[ISI][Medline]
Kristt DA, Waldman JV (1981) The origin of the acetylcholinesterase-rich afferents to layer IV of infant somatosensory cortex: a histochemical analysis following lesions. Anat Embryol 163:3141.[ISI][Medline]
Lebrand C, Cases O, Adelbrecht C, Doye A, Alvarez C, El Mestikawy S, Seif I, Gaspar P (1996) Transient uptake and storage of serotonin in developing thalamic neurons. Neuron 17:823835.[ISI][Medline]
Leslie MJ, Bennett-Clarke CA, Rhoades RW (1992) Serotonin 1B receptors form a transient vibrissa-related pattern in the primary somatosensory cortex of the developing rat. Dev Brain Res 69:143148.[ISI][Medline]
Levitt P, Harvey JA, Friedman E, Simansky K, Murphy EH (1997) New evidence for neurotransmitter influences on brain development. Trends Neurosci 20:269274.[ISI][Medline]
Lieske V, Bennett-Clarke CA, Rhoades RW (1999) Effects of serotonin on neurite outgrowth from thalamic neurons in vitro. Neuroscience 90: 967974.[ISI][Medline]
Lucas JJ, Yamamoto A, Scearce-Levie K, Saudou F, Hen R (1998) Absence of fenfluramine-induced anorexia and reduced c-fos induction in the hypothalamus and central amygdaloid complex of serotonin 1B receptor knock-out mice. J Neurosci 18:55375544.
Manjarrez GG, Chagoya GG, Hernandez J (1994) Early nutritional changes modify the kinetics and phosphorylation capacity of tryptophan-5-hydroxylase. Int J Dev Neurosci 12:695702.[ISI][Medline]
Manjarrez GG, Magdaleno VM, Chagoya GG, Hernandez J (1996) Nutritional recovery does not reverse the activation of brain serotonin synthesis in the ontogenetically malnourished rat. Int J Dev Neurosci 14:641648.[ISI][Medline]
Mansour-Robaey S, Mechawar N, Radja F, Beaulieu C, Descarries L (1998) Quantified distribution of serotonin transporter and receptors during the postnatal development of the rat barrel field cortex. Dev Brain Res 107:159163.[ISI][Medline]
Marin MC, De Tomas ME, Serres C, Mercuri O (1995) Protein-energy malnutrition during gestation and lactation in rats affects growth rate, brain development and essential fatty acid metabolism. J Nutr 125: 10171024.[ISI][Medline]
Miller FP, Cox RH Jr, Snodgrass WR, Maickel RP (1970) Comparative effects of p-chlorophenylalanine, p-chloroamphetamine and p-chloro- N-methylamphetamine on rat brain norepinephrine, serotonin and 5-hydroxyindole-3-acetic acid. Biochem Pharmacol 19:435442.[Medline]
Niitsu Y, Hamada S, Hamaguchi K, Mikuni M, Okado N (1995) Regulation of synapse density by 5-HT2A receptor agonist and antagonist in the spinal cord of chicken embryo. Neurosci Lett 195:159162.[ISI][Medline]
Okado N, Shibanoki S, Ishikawa K, Sako H (1989) Developmental changes in serotonin levels in the chick spinal cord and brain. Dev Brain Res 50:217223.[ISI][Medline]
Okado N, Cheng L, Tanatsugu Y, Hamada S, Hamaguchi K (1993) Synaptic loss following removal of serotoninergic fibers in newly hatched and adult chickens. J Neurobiol 24:687698.[ISI][Medline]
Osterheld-Haas MC, Van der Loos H, Hornung JP (1994) Monoaminergic afferents to cortex modulate structural plasticity in the barrelfield of the mouse. Dev Brain Res 77:189202.[ISI][Medline]
Persico AM, Calia E, Keller F (1997) Implants for sustained drug release over the somatosensory cortex of the newborn rat: a comparison of materials and surgical procedures. J Neurosci Methods 76:105113.[ISI][Medline]
Persico AM, Baldi A, Calia E, Moessner R, Lesch KP, Murphy DL, Keller F (1998) Alterations in neonatal barrel cortex and in the ageing brain of serotonin transporter knockout mice. Soc Neurosci Abs 24:1111.
Rhoades RW, Bennett-Clarke CA, Chiaia NL, White AF, Macdonald GJ, Haring JH, Jacquin MJ (1990) Development and lesion induced reorganization of the cortical representation of the rat's body surface as revealed by immunocytochemistry for serotonin. J Comp Neurol 293:190207.[ISI]
Rhoades RW, Bennett-Clarke CA, Shi MY, Mooney RD (1994) Effects of 5-HT on thalamocortical synaptic transmission in the developing rat. J Neurophysiol 72:24382450.
Rhoades RW, Chiaia NL, Lane RD, Bennett-Clarke CA (1998) Effect of activity blockade on changes in vibrissae-related patterns in the rat's primary somatosensory cortex induced by serotonin depletion. J Comp Neurol 402:276283.[ISI][Medline]
Rice FL (1995) Comparative aspects of barrel structure and development. In: Cerebral cortex, Vol. 11. The barrel cortex of rodents (Jones EG, Diamond IT, eds), pp. 176. New York: Plenum Press.
Riddle DR, Purves D (1995) Individual variation and lateral asimmetry of the rat primary somatosensory cortex. J Neurosci 15:41844195.[Abstract]
Riddle DR, Richards A, Zsuppan F, Purves D (1992) Growth of the rat somatic sensory cortex and its constituent parts during postnatal development. J Neurosci 12:30593524.
Robertson RT (1987) A morphogenic role for transiently expressed acetylcholinesterase in developing thalamocortical systems? Neurosci Lett 75:259264.[ISI][Medline]
Sikich L, Hickok JM, Todd RD (1990) 5HT1A receptors control neurite branching during development. Dev Brain Res 56:269274.[ISI][Medline]
Soto-Moyano R, Belmar J, Perez H, Ruiz S, Hernandez A (1995) Central noradrenergic hyperactivity early in life: a hypothesis on the origin of morpho-functional brain disorders induced by malnutrition. Biol Res 28:105111.[Medline]
Strominger RN, Woolsey TA (1987) Templates for locating the whiskers area in fresh flattened mouse and rat cortex. J Neurosci Methods 22:113118.[ISI][Medline]
Tabachnick BG, Fidell LS (1989) Using multivariate statistics. New York: Harper & Row.
Vongdokmai R (1980) Effect of protein malnutrition on development of mouse cortical barrels. J Comp Neurol 191:283294.[ISI][Medline]