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A tryptophan-deficient corn-based diet induces plastic responses in cerebellar cortex cells of rat offspring
Int. J. Devl Neuroscience 19 (2001) 447– 453 www.elsevier.nl/locate/ijdevneu
A tryptophan-deficient corn-based diet induces plastic responses in cerebellar cortex cells of rat offspring A.R. Del Angel-Meza a, L. Ramı´rez-Corte´s a, E. Olvera-Corte´s b, M.I. Pe´rez-Vega b, I. Gonza´lez-Burgos b,* a
Laboratorio de Nutricio´n Experimental, Centro de In6estigacio´n Biome´dica de Occidente, Instituto Mexicano del Seguro Social, Guadalajara, Jal., Mexico b Laboratorio de Psicobiologı´a, Centro de In6estigacio´n Biome´dica de Michoaca´n, Instituto Mexicano del Seguro Social, A.P. 7 -70, C.P. 58261, Morelia, Mich. Mexico Received 22 August 2000; received in revised form 27 October 2000; accepted 2 January 2001
Abstract Sprague–Dawley male rats, fed with a tryptophan-deficient and 8% protein corn-based diet were compared with a group of animals fed with 8% protein alone, and with a group fed with Chow Purina containing 23% protein. Retardation of Bergmann glial cell maturation and a concomitant retardation in granule cell migration were observed in the corn-fed group at 21 days. At 30 days of age, the dendrites of granule cells of both hypoproteic and corn-fed groups were larger than those of the Chow-fed animals. At 60 days of age, dendritic arborization of Purkinje cells was more profuse in both the hypoproteic and corn-fed rats compared with the Chow-fed group. This retardation in granule cell migration could be partially due to Bergmann glial cell immaturity. Consequently, several plastic and maybe compensatory events in both granule and Purkinje cells could have occurred, due to tryptophan deficiency resulting from the corn-based diet. © 2001 ISDN. Published by Elsevier Science Ltd. All rights reserved. Keywords: Corn-based diet; Hypoproteic diet; Tryptophan; Undernutrition
1. Introduction There is evidence that malnutrition can induce morphological and functional alterations in brain development. According to the life stage affected, this can occur by affecting the number of cells formed, by influencing cell migration, by delaying or blocking cell growth and differentiation, or by increasing cell death (Levitsky and Strump, 1995; Morgane et al., 1993). Neuronal and glial differentiation and synaptogenesis depend on the brain region involved and its critical developmental periods. Cerebellar maturation takes place postnatally, and it has been reported that the cerebellum is more sensitive to a wide range of adverse Abbre6iations: 5-HT, serotonin; ANOVA, analysis of variance; C, corn-based diet; Ch, control diet; H, hypoproteic diet; Try, L-tryptophan. * Corresponding author. Tel./Fax: + 52-43-241610. E-mail address: [email protected] (I. Gonza´lez-Burgos).
environmental factors than the rest of the brain during its early postnatal development (Barnes and Altman, 1973; Hillman and Chen, 1981; Shambaugh et al., 1996). It has been reported that neurogenesis, cell differentiation, migration and maturation, synaptogenesis, regression and programmed cell death could be affected by monoamines, because of their paracrine role in the regulation of cellular events in early life (Morgane et al., 1993; Soto-Moyano et al., 1998). During embryonic development, serotonin (5-hydroxytryptamine: 5-HT) seems to be involved in the processes of neuronal growth and differentiation. There is evidence from our own and other laboratories, pointing to specific changes in brain serotoninergic activity after early malnutrition (Del Angel-Meza et al., 1989; Fernstrom, 1981; Herna´ndez et al., 1989; Manjarre´z et al., 1994; Morgane et al., 1993). Serotonin concentration in the brain depends on the availability of L-tryptophan (Try). In experimental con-
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ditions using corn (which is Try-deficient) as a sole source of protein, a decrease in the synthesis, uptake and degradation of 5-HT have been observed, mainly in the rat cerebellum and brain stem (Beas-Za´ rate et al., 1988; Fernstrom, 1981). Protein restriction reduces the rates of cell differentiation, and results in impairments in cell migration and in synaptogenesis. In addition, a decrease of both brain 5-HT levels and serotoninergic activity takes place during brain development when there is Try deficiency in the diet. Thus, the aim of this study was to investigate the possible effects of Try deficiency on cerebellar cytoarchitecture using a corn diet.
2. Experimental procedures Adult female Sprague– Dawley rats were caged individually, and maintained under regular light– dark cycles of 12+ 12 h (07:00 – 19:00 h), an environmental humidity of 45–50%, a temperature of 229 2°C, and access to water and food ad libitum. The animals were divided into three groups. These were fed different diets: Chow-Purina-based 23% protein diet as a control (Ch); an 8% protein diet in a Chow-Purina base (hypoproteic: H); and a Try-deficient and 8% protein cornbased diet (C), according to a modification of Fernstrom and Hirsch’s model (Fernstrom and Hirsch, 1975). All three diets were isocaloric, and diets H and C were completed with vegetable oil, vitamins, minerals, and fiber content (Table 1). Diets were provided to the rats, commencing 5 weeks before mating with normal male rats. The feeding regimes were maintained until weaning of the pups and prolonged until the corresponding days of study. At 21, 30, or 60 days of age, 10 pups per group were weighed and administered ether as an anesthetic. They Table 1 Composition of diets (g/100 g diet) Components Corn meal Chow Purina Vegetable oil Dextrose Sucrose Dextrin Vitamin mixturea Mineral mixture R-Ha Non-nutritive fibera % Protein kcal/100 g
Chow-based
3. Results Hypoproteic
Corn-based
– 98.0 2.0 – – – – –
– 34.04 3.13 19.0 20.1 12.67 1.0 1.0
86.0 – 2.0 – – – 1.0 2.1
–
9.06
8.9
8.0 350.46
8.0 346.5
23.0 350.0
were perfused intracardially with 200 ml of a washing solution made up of phosphate buffer (pH 7.4, 0.1M), to which were added 1000 Ul/l of heparin as an anticoagulant and 1 g/l of procaine as a vasodilator. Thereafter, 200 ml of phosphate-buffered 4% formaldehyde was also perfused. Both solutions were perfused under 140 cm of water pressure (Feria-Velasco and Karnovsky, 1970). The brain was extracted by craniotomy and placed into a buffered solution of 4% formaldehyde for a period of at least 24 h. The whole brain and the cerebellum alone were weighed and evaluated in relation to body weight. Statistical analysis was carried out using analysis of variance (ANOVA) and Tukey tests. Two fragments of the cerebellar vermis were dissected out, starting from the midline. From the right tissue block, several 10-mm thick sagittal sections were obtained and stained with cresyl violet. The external granule cell layer’s migration pattern was evaluated in the anterior lobule. The left tissue block was processed by a modified Golgi method (Gonza´ lez-Burgos et al., 1992), and three well-impregnated granule and Purkinje cells per animal were studied. Both the area of the soma, as well as the length of the larger dendrite of the granule cells, were measured. Likewise, the area of the soma of the Purkinje cells was also measured. The dendritic arborization of these neurons was quantified by the Sholl method (Sholl, 1953), using five concentric rings from the soma, 25 mm apart from each other (Fig. 1). Finally, the cytoarchitectonic characteristics of the Bergmann glial cells were evaluated. All the cytoarchitectonic measurements were made with an image analyzer (Zeiss Image 3.0; Carl Zeiss, Inc.; Thornwood, NY). Averages of the corresponding values of the three neurons studied per rat were in turn averaged, and ANOVA and Tukey tests were used for statistical comparisons.
a Tecklad (cat. No. 170760, 40060, and 160390; respectively. Madison, Wisconsin. USA).
The three groups did not differ significantly in the quantity of food eaten (data not shown). At 21 days, the mean body weight of the C group was less than that of both Ch and H groups, while the H group was less than the Ch animals. On the other hand, the brain and cerebellar weights of the H group were not different compared with those of Ch animals. However, the C rats had lower brain and cerebellar weights compared with both Ch and H groups at the same age. At 30 days, both body and brain weights showed significant differences among all three groups. There were no differences in the cerebellar weight of the H group when compared with the Ch and C animals, while the cerebellum of the C animals was different only in respect to that of the Ch rats. At 60 days, body, brain
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Fig. 1. Composition of the cytoarchitectural measurements made both on cerebellar granule cells (A1: area of cell soma; A2: dendritic length), and on Purkinje cells (B1: area of cell soma; B2: dendritic arborization measured by the Sholl method). Scale bars: A, 30 mm; B, 50 mm.
and cerebellar weights were different between the three groups (Table 2). Morphological study of the cerebellar cortex revealed that, at 21 days, cells of the external granule layer were still evident in group C, while in both H and Ch groups, granule cell migration was complete. At 30 days of age, there was no external granule layer in any of the three groups (Fig. 2). The cytoarchitectonic pattern of the Bergmann glial cells of group C at 21 days showed the presence of more basal thick filopodia, when compared with H Bergmann glia. In addition, Bergmann cells in group C showed greater numbers of pial-directed processes and some of these were shorter compared with both H and Ch groups. Group Ch’s Bergmann cells did not show basal filopodia and their radial processes had fewer cytoplasmic excrescences than those of both H and C groups. Furthermore, the cytoplasmic excrescences of H and C groups were more voluminous than those from Ch group. At 30 days of age, the Bergmann glia of all three groups studied showed similar cytoarchitecture (Fig. 3). The area of the soma of granule cells showed differences only at 30 days. Group H showed less cell soma area when compared with group Ch (Fig. 4). Likewise, at 30 days of age the granule cell dendrites of both H and C groups were significantly larger than those of the Ch group (Fig. 5).
The area of the soma of Purkinje cells showed differences only at 21 and 60 days. This was less in group H compared with Ch at 21and 60 days of age (Fig. 6). As to dendritic arborization, there were significant differences only at 21 and 60 days of age too. At 21 days, the intersections of Purkinje cell dendrites with concentric
Table 2 Body, brain, and cerebellar weights (g) of the three groups studied
Body weight Chow-based Hypoproteic Corn-based Brain weight Chow-based Hypoproteic Corn-based Cerebellar weight Chow-based Hypoproteic Corn-based a
21-day
30-day
60-day
56.1 9 2.8 21.3 92.0a 24.5 91.3b,c
85.4 93.0 31.0 92.6a 27.1 9 2.2b,c
185.8 95.7 72.6 9 4.2a 35.4 9 4.0b,c
1.41 9 0.03 1.43 9 0.02 1.06 9 0.03b,c
1.89 9 0.05 1.58 9 0.04a 1.25 90.03b,c
1.92 9 0.01 1.82 9 0.06a 1.49 9 0.07b,c
0.17 90.005 0.17 9 0.005 0.11 9 0.003b,c
0.23 90.02 0.22 90.02 0.20 90.01b
0.24 90.01 0.23 9 0.01a 0.20 9 0.008b,c
Chow-based versus hypoproteic. Chow-based versus corn-based. c Hypoproteic versus corn-based. Means 9 SD, PB0.01. b
450
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Fig. 2. Comparative micrographs of the 21-day-old cerebellar cortex of chow- (A1), hypoproteic- (A2), and corn-fed (A3) rats. Note the presence of external granule layer cells (arrows) in the upper molecular layer in the latter (m). At 30 days, the external granule layer cells of the chow- (B1), hypoproteic- (B2), and xorn-fed (B3) animals had migrated completely to constitute the granule layer (g). Cresyl violet; 40 × .
rings numbers two and five were less in H group compared with both Ch and C groups. However, the number of intersections in ring number four of the C group was greater compared only with group H. At 60 days, dendritic arborization of Purkinje cells from C group was more profuse in rings one and two, while rings two, three and four of H group showed more intersections compared with the Ch group of animals (Table 3).
4. Discussion The body mass of rats in group H was lower at all three ages studied in comparison with the Ch group. However, brain weight was affected only at 30 and 60 days of age, and cerebellar weight was less only at 60 days. On the other hand, the corn-fed animals showed lower body, brain and cerebellar weights than both Ch and H groups at all three ages studied. It is well known that hypoproteic undernutrition leads to low body and brain weights (Del Angel-Meza et al., 1989). Moreover, Try is strongly involved in growth hormone activity (Rivest, 1991), as well as in glioblast proliferation, neurogenesis and cell migration in the developing cerebellum (Bishop et al., 1985; Lauder and Krebs, 1978; Lauder et al., 1981, 1983). This could be closely associated with the stronger effects observed in these parameters in the corn-fed animals in relation to the H group, when compared with normal rats. In the corn-fed group of animals, a possible overproduction and delayed cell death, or a delay in the
external granule cells migration could have occurred. The latter seems more likely because of the abnormal development of Bergmann glia observed in this same group of animals. A close relationship between Bergmann glial cell maturation and granule cell migration is well known (Gregory et al., 1988; Hager et al., 1995; Hatten and Mason, 1990). Furthermore, it has been shown, in normal rats, that the pial-directed processes of Bergmann glial cells decrease gradually from postnatal days 8–12, that cytoplasmic excrescences increase in number (Shiga et al., 1983), and that basal filopodia disappear between 15 and 19 days of age (Das, 1976). Bergmann glial cells of hypoproteic-fed and corn-fed animals showed an abnormal ‘hypergrowth’ of cytoplasmic excrescences in all the pial-directed fibers, which were present in greater numbers than in the Ch group. In addition, in the corn-fed rats, Bergmann glial cells showed a persistence of basal filopodia, as well as shorter pial-directed fibers. Thus, as glioblast proliferation and development are affected by 5-HT availability (Lauder et al., 1983), and as undernutrition leads to a delayed persistence of the external granule layer (Del Angel-Meza et al., 1984; Sima and Peerson, 1975), a close parallel between the effects observed in both Bergmann glia and in the external granule layer migration may have occurred due to feeding on corn. Analysis of granule cell cytoarchitecture revealed that the area of the soma of H neurons was smaller than that of Ch cells at 30 days of age, along with an enlargement of their dendrites. This latter feature was also observed in corn-fed animals. It is known that at
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Fig. 5. Graph corresponding to dendritic length of the granule cells of each of the three groups of animals. Means 9SD. *: chow-based versus hypoproteic; + : chow-based versus corn-fed. P B0.05.
least some mossy fibers are of serotoninergic nature (Chan-Palay, 1975). Thus, starting from low levels of 5-HT, enlargement of granule cell dendrites could be interpreted as a plastic response. However, why this occurred only at 30 days cannot be elucidated from the present work. Therefore, both cellular mechanisms as well as the functional significance of this finding must be further investigated.
At 21 days, the area of Purkinje cell soma of H group was smaller than both the Ch and C groups of animals. Likewise, dendritic arborization of H Purkinje neurons was less than that of the Ch and C groups, at the same age. This finding is in agreement with previous reports which showed both the growth of Purkinje cell soma and its dendritic tree in normal conditions (Altman and Winfree, 1977), as well as retarded outgrowth of the dendritic tree of Purkinje cells, caused by pre- and postnatal undernutrition (Sima and Peerson, 1975). At 60 days of age, the area of the Purkinje cell soma of the H group was also less than for the other two groups of animals. However, dendritic arborization of both H and C groups was observed to be more profuse than for Ch rats. The dendritic arborization of experimental Purkinje cells was also more profuse in the segments proximal to the soma, whereas in the H group, this took place in the distal segments. Since the area of the soma was smaller only in the H animals, this could be related to some plastic mechanism, leading to Purkinje
Fig. 4. Comparison of the area of the granule cell soma, between the three groups studied. Means 9 SD. *: chow-based versus hypoproteic. P B0.05.
Fig. 6. Area of the Purkinje cell soma of each group of animals studied. Means9 SD. *: chow-based versus hypoproteic. PB 0.05.
Fig. 3. Camera lucida drawings of Bergmann glial cells of the chow(A1), hypoproteic- (A2), and corn-fed (A3) rats. Both hypoproteic- and corn-fed animals showed Bergmann cells with more voluminous cytoplasmic excrescences (asterisks) than those seen in normal rats. Furthermore, Bergman glial cells of the corn-fed animals also showed the persistence of basal filopodia (arrowhead), as well as shorter pial-directed radial processes than those seen in both hypoproteic- and chow-fed animals (arrows). At 30 days of age, morphological features of the Bergmann cells of hypoproteic- (B2) and corn-fed (B3) rats were similar in comparison with those of the chow-fed (B1) animals.
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Table 3 Number of intersections of the dendrites of Purkinje cells with each of five concentric rings 25 mm apart from each other
21-Day Chow-based Hypoproteic Corn-based 30-Day Chow-based Hypoproteic Corn-based 60-Day Chow-based Hypoproteic Corn-based
1st
2nd
3rd
4th
5th
10.69 0.8 8.29 0.9 10.49 0.8
21.09 1.0 16.59 1.4a 22.69 1.3c
22.3 9 1.5 17.6 9 2.3 22.4 9 2.2
16.4 9 2.2 9.0 91.5 17.2 92.8c
9.3 9 1.8 2.2 9 0.9a 12.2 9 2.2c
10.290.7 11.59 1.0 12.691.1
20.99 1.4 23.59 1.8 24.99 2.0
25.3 9 1.8 24.9 9 2.5 27.7 9 3.3
24.7 9 2.2 19.69 4.1 20.8 9 1.9
14.4 9 2.6 12.8 9 3.9 10.0 91.8
11.390.6 15.99 1.5 17.4 9 2.0b
24.69 1.3 40.1 9 2.0a 37.5 93.5b
32.3 9 2.5 49.8 9 3.0a 41.4 9 3.2
31.9 9 2.5 44.4 9 3.4a 40.2 92.8
25.5 9 2.6 23.9 93.0 23.4 9 3.1
a
Chow-based versus hypoproteic. Chow-based versus corn-based. c Hypoproteic versus corn-based. Means 9 SD, PB0.05. b
cells increasing their area of synaptic contact, in both hypoproteic- and corn-fed groups of animals, in adulthood. It has been reported that cerebellar serotoninergic neurons terminate as mossy fibers in glomeruli; as parallel-like fibers on dendrites of basket and stellate cells (Chan-Palay, 1975); as climbing fibers on dendrites of Purkinje cells (Beas-Za´ rate et al., 1988); and as a diffuse system in both granule and molecular layers (Chan-Palay, 1975) of the immature cerebellum (Matthiessen et al., 1993). On the other hand, it is known that 5-HT modulates fast excitatory synaptic activity in the cerebellar cortex (Lu and Larson-Prior, 1996; Maura et al., 1995). Moreover, it enhances the GABA-mediated inhibitory postsynaptic currents in Purkinje cells (Mitoma et al., 1994). Thus, cerebellar serotoninergic activity plays an important role both in the integration of incoming sensory signals by granule and Purkinje cells, as well as in the modulation of the output from the cerebellar cortex through presynaptic facilitation. In the latter context, the present findings suggest that the low levels of cerebellar 5-HT induced by corn feeding, may have led to retardation in the maturation of cerebellar cortex. This in turn could trigger some plastic neural responses, to compensate the possible alterations in cerebellar cortical activity. References Altman, J., Winfree, A.T., 1977. Postnatal development of the cerebellar cortex in the rat. V. Spatial organization of Purkinje cell perikarya. J. Comp. Neurol. 171, 1 – 16. Barnes, D., Altman, J., 1973. Effects of different schedules of early undernutrition on the preweaning growth of the rat cerebellum. Exp. Neurol. 38, 406 –419. Beas-Za´ rate, C., Del Angel-Meza, A.R., Morales-Villagra´ n, A., Feria-Velasco, A., 1988. Serotonin uptake in the central nervous system of rats fed a corn diet. Comp. Biochem. Physiol. 89C, 173– 177.
Bishop, G.A., Ho, R.H., King, J.S., 1985. An immunohistochemical study of serotonin development in the opossum cerebellum. Anat. Embryol. 171, 325 – 338. Chan-Palay, V., 1975. Fine structure of labelled axons in the cerebellar cortex and nuclei of rodents and primates after intraventricular infusions with tritiated serotonin. Anat. Embriol. 148, 235 – 265. Das, G.D., 1976. Differentiation of Bergmann glia cells in the cerebellum: a Golgi study. Brain Res. 110, 199 – 213. Del Angel-Meza, A.R., Beas-Za´ rate, C., Morales-Villagra´ n, A., 1989. Effects of corn-fed and protein restriction on rat cerebellum and brain stem maturation. Nutr. Rep. Int. 40, 1199 – 1206. Del Angel-Meza, A.R., Tapia-Arizmendi, G., Feria-Velasco, A., 1984. Effects of food restriction during lactation on postnatal development of rat cerebellum. Correlative biochemical and structural study. Nutr. Rep. Int. 30, 95 – 109. Feria-Velasco, A., Karnovsky, M.J., 1970. Preservacio´ n o´ ptima del sistema nervioso central por perfusio´ n con glutaraldehı´do para estudio ultraestructural. Arch. Inv. Med. 1, 201 – 220. Fernstrom, J.D., 1981. Effects of the diet and other metabolic phenomena on brain tryptophan uptake and serotonin synthesis. Ann. Rev. Med. 23, 413 – 425. Fernstrom, J.D., Hirsch, M.J., 1975. Rapid depletion of brain serotonin in malnourished corn-fed rats following L-tryptophan injection. Life Sci. 17, 445 – 452. Gonza´ lez-Burgos, I., Tapia-Arizmendi, G., Feria-Velasco, A., 1992. Golgi method without osmium tetroxide for the study of the central nervous system. Biotech. Histochem. 67, 288 – 296. Gregory, W.A., Edmondson, J.C., Hatten, M.E., Mason, C.A., 1988. Cytology and neuron-glial apposition of migrating cerebellar granule cells in vitro. J. Neurosci. 8, 1728 – 1738. Hager, G., Dodt, H.U., Zieglgansberger, W., Liesi, P., 1995. Novel forms of neuronal migration in the rat cerebellum. Neurosci. Res. 40, 207 – 219. Hatten, M.E., Mason, C.A., 1990. Mechanisms of glial-guided neuronal migration in vitro and in vivo. Experientia 46, 907 –916. Herna´ ndez, R.J., Manjarre´ z, G.G., Chagoya, G., 1989. Newborn humans and rats malnourished in utero: free plasma L-tryptophan, neutral amino acids and brain serotonin synthesis. Brain Res. 488, 1 – 13. Hillman, D.E., Chen, S., 1981. Vulnerability of cerebellar development in malnutrition. I. Quantitation of layer volume and neuron numbers. Neuroscience 6, 1249 – 1262. Lauder, J.M., Krebs, H., 1978. Serotonin as a differentiation signal in early neurogenesis. Dev. Neurosci. 1, 15 – 30.
A.R. Del Angel-Meza et al. / Int. J. De6l Neuroscience 19 (2001) 447–453 Lauder, J.M., Wallace, J.A., Krebs, H., 1981. Roles for serotonin in neuroembryogenesis. Adv. Exp. Med. Biol. 133, 477 –506. Lauder, J.M., Wallace, J.A., Wilkie, M.B., Krebs, H., 1983. Roles for serotonin in neurogenesis. Monogr. Neural Sci. 9, 3 –10. Levitsky, D.A., Strump, B.J., 1995. Malnutrition and the brain: changing concepts, changing concerns. J. Nutr. 125, 2212S – 2220S. Lu, H., Larson-Prior, L.J., 1996. Serotonergic modulation of evoked responses in turtle cerebellar Purkinje cells. J. Neurophysiol. 76, 3102 – 3113. Manjarre´ z, G.G., Chagoya, G., Herna´ ndez, R.J., 1994. Early nutritional changes modify the kinetics and phosphorylation capacity of tryptophan-5-hydroxylase. Int. J. Devl. Neurosci. 12, 695 – 702. Matthiessen, L., Kia, H.K., Daval, G., Riad, M., Hamon, M., Verge, D., 1993. Immunocytochemical localization of 5-HT1A receptors in the rat immature cerebellum. Neuroreport 4, 763 –766. Maura, G., Guadagnin, G., Raiteri, M., 1995. Low nanomolar serotonin inhibits the glutamate receptor/nitric oxide/cyclic GMP pathway in slices from adult rat cerebellum. Neuroscience 68, 455 – 463. Mitoma, H., Kobayashi, T., Song, S.Y., Konishi, S., 1994. Enhancement by serotonin of GABA-mediated inhibitory synaptic currents in rat cerebellar Purkinje cells. Neurosci. Lett. 173, 127 – 130. Morgane, P.J., Austin-Lafrance, R., Bronzino, J., Tonkiss, J., Dı´azCintra, S., Cintra, L., Kemper, T., Galler, R.J., 1993. Prenatal
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A tryptophan-deficient corn-based diet induces plastic responses in cerebellar cortex cells of rat offspring A.R. Del Angel-Meza a, L. Ramı´rez-Corte´s a, E. Olvera-Corte´s b, M.I. Pe´rez-Vega b, I. Gonza´lez-Burgos b,* a
Laboratorio de Nutricio´n Experimental, Centro de In6estigacio´n Biome´dica de Occidente, Instituto Mexicano del Seguro Social, Guadalajara, Jal., Mexico b Laboratorio de Psicobiologı´a, Centro de In6estigacio´n Biome´dica de Michoaca´n, Instituto Mexicano del Seguro Social, A.P. 7 -70, C.P. 58261, Morelia, Mich. Mexico Received 22 August 2000; received in revised form 27 October 2000; accepted 2 January 2001
Abstract Sprague–Dawley male rats, fed with a tryptophan-deficient and 8% protein corn-based diet were compared with a group of animals fed with 8% protein alone, and with a group fed with Chow Purina containing 23% protein. Retardation of Bergmann glial cell maturation and a concomitant retardation in granule cell migration were observed in the corn-fed group at 21 days. At 30 days of age, the dendrites of granule cells of both hypoproteic and corn-fed groups were larger than those of the Chow-fed animals. At 60 days of age, dendritic arborization of Purkinje cells was more profuse in both the hypoproteic and corn-fed rats compared with the Chow-fed group. This retardation in granule cell migration could be partially due to Bergmann glial cell immaturity. Consequently, several plastic and maybe compensatory events in both granule and Purkinje cells could have occurred, due to tryptophan deficiency resulting from the corn-based diet. © 2001 ISDN. Published by Elsevier Science Ltd. All rights reserved. Keywords: Corn-based diet; Hypoproteic diet; Tryptophan; Undernutrition
1. Introduction There is evidence that malnutrition can induce morphological and functional alterations in brain development. According to the life stage affected, this can occur by affecting the number of cells formed, by influencing cell migration, by delaying or blocking cell growth and differentiation, or by increasing cell death (Levitsky and Strump, 1995; Morgane et al., 1993). Neuronal and glial differentiation and synaptogenesis depend on the brain region involved and its critical developmental periods. Cerebellar maturation takes place postnatally, and it has been reported that the cerebellum is more sensitive to a wide range of adverse Abbre6iations: 5-HT, serotonin; ANOVA, analysis of variance; C, corn-based diet; Ch, control diet; H, hypoproteic diet; Try, L-tryptophan. * Corresponding author. Tel./Fax: + 52-43-241610. E-mail address: [email protected] (I. Gonza´lez-Burgos).
environmental factors than the rest of the brain during its early postnatal development (Barnes and Altman, 1973; Hillman and Chen, 1981; Shambaugh et al., 1996). It has been reported that neurogenesis, cell differentiation, migration and maturation, synaptogenesis, regression and programmed cell death could be affected by monoamines, because of their paracrine role in the regulation of cellular events in early life (Morgane et al., 1993; Soto-Moyano et al., 1998). During embryonic development, serotonin (5-hydroxytryptamine: 5-HT) seems to be involved in the processes of neuronal growth and differentiation. There is evidence from our own and other laboratories, pointing to specific changes in brain serotoninergic activity after early malnutrition (Del Angel-Meza et al., 1989; Fernstrom, 1981; Herna´ndez et al., 1989; Manjarre´z et al., 1994; Morgane et al., 1993). Serotonin concentration in the brain depends on the availability of L-tryptophan (Try). In experimental con-
0736-5748/01/$ © 2001 ISDN. Published by Elsevier Science Ltd. All rights reserved. PII: S 0 7 3 6 - 5 7 4 8 ( 0 1 ) 0 0 0 0 4 - 1
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ditions using corn (which is Try-deficient) as a sole source of protein, a decrease in the synthesis, uptake and degradation of 5-HT have been observed, mainly in the rat cerebellum and brain stem (Beas-Za´ rate et al., 1988; Fernstrom, 1981). Protein restriction reduces the rates of cell differentiation, and results in impairments in cell migration and in synaptogenesis. In addition, a decrease of both brain 5-HT levels and serotoninergic activity takes place during brain development when there is Try deficiency in the diet. Thus, the aim of this study was to investigate the possible effects of Try deficiency on cerebellar cytoarchitecture using a corn diet.
2. Experimental procedures Adult female Sprague– Dawley rats were caged individually, and maintained under regular light– dark cycles of 12+ 12 h (07:00 – 19:00 h), an environmental humidity of 45–50%, a temperature of 229 2°C, and access to water and food ad libitum. The animals were divided into three groups. These were fed different diets: Chow-Purina-based 23% protein diet as a control (Ch); an 8% protein diet in a Chow-Purina base (hypoproteic: H); and a Try-deficient and 8% protein cornbased diet (C), according to a modification of Fernstrom and Hirsch’s model (Fernstrom and Hirsch, 1975). All three diets were isocaloric, and diets H and C were completed with vegetable oil, vitamins, minerals, and fiber content (Table 1). Diets were provided to the rats, commencing 5 weeks before mating with normal male rats. The feeding regimes were maintained until weaning of the pups and prolonged until the corresponding days of study. At 21, 30, or 60 days of age, 10 pups per group were weighed and administered ether as an anesthetic. They Table 1 Composition of diets (g/100 g diet) Components Corn meal Chow Purina Vegetable oil Dextrose Sucrose Dextrin Vitamin mixturea Mineral mixture R-Ha Non-nutritive fibera % Protein kcal/100 g
Chow-based
3. Results Hypoproteic
Corn-based
– 98.0 2.0 – – – – –
– 34.04 3.13 19.0 20.1 12.67 1.0 1.0
86.0 – 2.0 – – – 1.0 2.1
–
9.06
8.9
8.0 350.46
8.0 346.5
23.0 350.0
were perfused intracardially with 200 ml of a washing solution made up of phosphate buffer (pH 7.4, 0.1M), to which were added 1000 Ul/l of heparin as an anticoagulant and 1 g/l of procaine as a vasodilator. Thereafter, 200 ml of phosphate-buffered 4% formaldehyde was also perfused. Both solutions were perfused under 140 cm of water pressure (Feria-Velasco and Karnovsky, 1970). The brain was extracted by craniotomy and placed into a buffered solution of 4% formaldehyde for a period of at least 24 h. The whole brain and the cerebellum alone were weighed and evaluated in relation to body weight. Statistical analysis was carried out using analysis of variance (ANOVA) and Tukey tests. Two fragments of the cerebellar vermis were dissected out, starting from the midline. From the right tissue block, several 10-mm thick sagittal sections were obtained and stained with cresyl violet. The external granule cell layer’s migration pattern was evaluated in the anterior lobule. The left tissue block was processed by a modified Golgi method (Gonza´ lez-Burgos et al., 1992), and three well-impregnated granule and Purkinje cells per animal were studied. Both the area of the soma, as well as the length of the larger dendrite of the granule cells, were measured. Likewise, the area of the soma of the Purkinje cells was also measured. The dendritic arborization of these neurons was quantified by the Sholl method (Sholl, 1953), using five concentric rings from the soma, 25 mm apart from each other (Fig. 1). Finally, the cytoarchitectonic characteristics of the Bergmann glial cells were evaluated. All the cytoarchitectonic measurements were made with an image analyzer (Zeiss Image 3.0; Carl Zeiss, Inc.; Thornwood, NY). Averages of the corresponding values of the three neurons studied per rat were in turn averaged, and ANOVA and Tukey tests were used for statistical comparisons.
a Tecklad (cat. No. 170760, 40060, and 160390; respectively. Madison, Wisconsin. USA).
The three groups did not differ significantly in the quantity of food eaten (data not shown). At 21 days, the mean body weight of the C group was less than that of both Ch and H groups, while the H group was less than the Ch animals. On the other hand, the brain and cerebellar weights of the H group were not different compared with those of Ch animals. However, the C rats had lower brain and cerebellar weights compared with both Ch and H groups at the same age. At 30 days, both body and brain weights showed significant differences among all three groups. There were no differences in the cerebellar weight of the H group when compared with the Ch and C animals, while the cerebellum of the C animals was different only in respect to that of the Ch rats. At 60 days, body, brain
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Fig. 1. Composition of the cytoarchitectural measurements made both on cerebellar granule cells (A1: area of cell soma; A2: dendritic length), and on Purkinje cells (B1: area of cell soma; B2: dendritic arborization measured by the Sholl method). Scale bars: A, 30 mm; B, 50 mm.
and cerebellar weights were different between the three groups (Table 2). Morphological study of the cerebellar cortex revealed that, at 21 days, cells of the external granule layer were still evident in group C, while in both H and Ch groups, granule cell migration was complete. At 30 days of age, there was no external granule layer in any of the three groups (Fig. 2). The cytoarchitectonic pattern of the Bergmann glial cells of group C at 21 days showed the presence of more basal thick filopodia, when compared with H Bergmann glia. In addition, Bergmann cells in group C showed greater numbers of pial-directed processes and some of these were shorter compared with both H and Ch groups. Group Ch’s Bergmann cells did not show basal filopodia and their radial processes had fewer cytoplasmic excrescences than those of both H and C groups. Furthermore, the cytoplasmic excrescences of H and C groups were more voluminous than those from Ch group. At 30 days of age, the Bergmann glia of all three groups studied showed similar cytoarchitecture (Fig. 3). The area of the soma of granule cells showed differences only at 30 days. Group H showed less cell soma area when compared with group Ch (Fig. 4). Likewise, at 30 days of age the granule cell dendrites of both H and C groups were significantly larger than those of the Ch group (Fig. 5).
The area of the soma of Purkinje cells showed differences only at 21 and 60 days. This was less in group H compared with Ch at 21and 60 days of age (Fig. 6). As to dendritic arborization, there were significant differences only at 21 and 60 days of age too. At 21 days, the intersections of Purkinje cell dendrites with concentric
Table 2 Body, brain, and cerebellar weights (g) of the three groups studied
Body weight Chow-based Hypoproteic Corn-based Brain weight Chow-based Hypoproteic Corn-based Cerebellar weight Chow-based Hypoproteic Corn-based a
21-day
30-day
60-day
56.1 9 2.8 21.3 92.0a 24.5 91.3b,c
85.4 93.0 31.0 92.6a 27.1 9 2.2b,c
185.8 95.7 72.6 9 4.2a 35.4 9 4.0b,c
1.41 9 0.03 1.43 9 0.02 1.06 9 0.03b,c
1.89 9 0.05 1.58 9 0.04a 1.25 90.03b,c
1.92 9 0.01 1.82 9 0.06a 1.49 9 0.07b,c
0.17 90.005 0.17 9 0.005 0.11 9 0.003b,c
0.23 90.02 0.22 90.02 0.20 90.01b
0.24 90.01 0.23 9 0.01a 0.20 9 0.008b,c
Chow-based versus hypoproteic. Chow-based versus corn-based. c Hypoproteic versus corn-based. Means 9 SD, PB0.01. b
450
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Fig. 2. Comparative micrographs of the 21-day-old cerebellar cortex of chow- (A1), hypoproteic- (A2), and corn-fed (A3) rats. Note the presence of external granule layer cells (arrows) in the upper molecular layer in the latter (m). At 30 days, the external granule layer cells of the chow- (B1), hypoproteic- (B2), and xorn-fed (B3) animals had migrated completely to constitute the granule layer (g). Cresyl violet; 40 × .
rings numbers two and five were less in H group compared with both Ch and C groups. However, the number of intersections in ring number four of the C group was greater compared only with group H. At 60 days, dendritic arborization of Purkinje cells from C group was more profuse in rings one and two, while rings two, three and four of H group showed more intersections compared with the Ch group of animals (Table 3).
4. Discussion The body mass of rats in group H was lower at all three ages studied in comparison with the Ch group. However, brain weight was affected only at 30 and 60 days of age, and cerebellar weight was less only at 60 days. On the other hand, the corn-fed animals showed lower body, brain and cerebellar weights than both Ch and H groups at all three ages studied. It is well known that hypoproteic undernutrition leads to low body and brain weights (Del Angel-Meza et al., 1989). Moreover, Try is strongly involved in growth hormone activity (Rivest, 1991), as well as in glioblast proliferation, neurogenesis and cell migration in the developing cerebellum (Bishop et al., 1985; Lauder and Krebs, 1978; Lauder et al., 1981, 1983). This could be closely associated with the stronger effects observed in these parameters in the corn-fed animals in relation to the H group, when compared with normal rats. In the corn-fed group of animals, a possible overproduction and delayed cell death, or a delay in the
external granule cells migration could have occurred. The latter seems more likely because of the abnormal development of Bergmann glia observed in this same group of animals. A close relationship between Bergmann glial cell maturation and granule cell migration is well known (Gregory et al., 1988; Hager et al., 1995; Hatten and Mason, 1990). Furthermore, it has been shown, in normal rats, that the pial-directed processes of Bergmann glial cells decrease gradually from postnatal days 8–12, that cytoplasmic excrescences increase in number (Shiga et al., 1983), and that basal filopodia disappear between 15 and 19 days of age (Das, 1976). Bergmann glial cells of hypoproteic-fed and corn-fed animals showed an abnormal ‘hypergrowth’ of cytoplasmic excrescences in all the pial-directed fibers, which were present in greater numbers than in the Ch group. In addition, in the corn-fed rats, Bergmann glial cells showed a persistence of basal filopodia, as well as shorter pial-directed fibers. Thus, as glioblast proliferation and development are affected by 5-HT availability (Lauder et al., 1983), and as undernutrition leads to a delayed persistence of the external granule layer (Del Angel-Meza et al., 1984; Sima and Peerson, 1975), a close parallel between the effects observed in both Bergmann glia and in the external granule layer migration may have occurred due to feeding on corn. Analysis of granule cell cytoarchitecture revealed that the area of the soma of H neurons was smaller than that of Ch cells at 30 days of age, along with an enlargement of their dendrites. This latter feature was also observed in corn-fed animals. It is known that at
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Fig. 5. Graph corresponding to dendritic length of the granule cells of each of the three groups of animals. Means 9SD. *: chow-based versus hypoproteic; + : chow-based versus corn-fed. P B0.05.
least some mossy fibers are of serotoninergic nature (Chan-Palay, 1975). Thus, starting from low levels of 5-HT, enlargement of granule cell dendrites could be interpreted as a plastic response. However, why this occurred only at 30 days cannot be elucidated from the present work. Therefore, both cellular mechanisms as well as the functional significance of this finding must be further investigated.
At 21 days, the area of Purkinje cell soma of H group was smaller than both the Ch and C groups of animals. Likewise, dendritic arborization of H Purkinje neurons was less than that of the Ch and C groups, at the same age. This finding is in agreement with previous reports which showed both the growth of Purkinje cell soma and its dendritic tree in normal conditions (Altman and Winfree, 1977), as well as retarded outgrowth of the dendritic tree of Purkinje cells, caused by pre- and postnatal undernutrition (Sima and Peerson, 1975). At 60 days of age, the area of the Purkinje cell soma of the H group was also less than for the other two groups of animals. However, dendritic arborization of both H and C groups was observed to be more profuse than for Ch rats. The dendritic arborization of experimental Purkinje cells was also more profuse in the segments proximal to the soma, whereas in the H group, this took place in the distal segments. Since the area of the soma was smaller only in the H animals, this could be related to some plastic mechanism, leading to Purkinje
Fig. 4. Comparison of the area of the granule cell soma, between the three groups studied. Means 9 SD. *: chow-based versus hypoproteic. P B0.05.
Fig. 6. Area of the Purkinje cell soma of each group of animals studied. Means9 SD. *: chow-based versus hypoproteic. PB 0.05.
Fig. 3. Camera lucida drawings of Bergmann glial cells of the chow(A1), hypoproteic- (A2), and corn-fed (A3) rats. Both hypoproteic- and corn-fed animals showed Bergmann cells with more voluminous cytoplasmic excrescences (asterisks) than those seen in normal rats. Furthermore, Bergman glial cells of the corn-fed animals also showed the persistence of basal filopodia (arrowhead), as well as shorter pial-directed radial processes than those seen in both hypoproteic- and chow-fed animals (arrows). At 30 days of age, morphological features of the Bergmann cells of hypoproteic- (B2) and corn-fed (B3) rats were similar in comparison with those of the chow-fed (B1) animals.
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Table 3 Number of intersections of the dendrites of Purkinje cells with each of five concentric rings 25 mm apart from each other
21-Day Chow-based Hypoproteic Corn-based 30-Day Chow-based Hypoproteic Corn-based 60-Day Chow-based Hypoproteic Corn-based
1st
2nd
3rd
4th
5th
10.69 0.8 8.29 0.9 10.49 0.8
21.09 1.0 16.59 1.4a 22.69 1.3c
22.3 9 1.5 17.6 9 2.3 22.4 9 2.2
16.4 9 2.2 9.0 91.5 17.2 92.8c
9.3 9 1.8 2.2 9 0.9a 12.2 9 2.2c
10.290.7 11.59 1.0 12.691.1
20.99 1.4 23.59 1.8 24.99 2.0
25.3 9 1.8 24.9 9 2.5 27.7 9 3.3
24.7 9 2.2 19.69 4.1 20.8 9 1.9
14.4 9 2.6 12.8 9 3.9 10.0 91.8
11.390.6 15.99 1.5 17.4 9 2.0b
24.69 1.3 40.1 9 2.0a 37.5 93.5b
32.3 9 2.5 49.8 9 3.0a 41.4 9 3.2
31.9 9 2.5 44.4 9 3.4a 40.2 92.8
25.5 9 2.6 23.9 93.0 23.4 9 3.1
a
Chow-based versus hypoproteic. Chow-based versus corn-based. c Hypoproteic versus corn-based. Means 9 SD, PB0.05. b
cells increasing their area of synaptic contact, in both hypoproteic- and corn-fed groups of animals, in adulthood. It has been reported that cerebellar serotoninergic neurons terminate as mossy fibers in glomeruli; as parallel-like fibers on dendrites of basket and stellate cells (Chan-Palay, 1975); as climbing fibers on dendrites of Purkinje cells (Beas-Za´ rate et al., 1988); and as a diffuse system in both granule and molecular layers (Chan-Palay, 1975) of the immature cerebellum (Matthiessen et al., 1993). On the other hand, it is known that 5-HT modulates fast excitatory synaptic activity in the cerebellar cortex (Lu and Larson-Prior, 1996; Maura et al., 1995). Moreover, it enhances the GABA-mediated inhibitory postsynaptic currents in Purkinje cells (Mitoma et al., 1994). Thus, cerebellar serotoninergic activity plays an important role both in the integration of incoming sensory signals by granule and Purkinje cells, as well as in the modulation of the output from the cerebellar cortex through presynaptic facilitation. In the latter context, the present findings suggest that the low levels of cerebellar 5-HT induced by corn feeding, may have led to retardation in the maturation of cerebellar cortex. This in turn could trigger some plastic neural responses, to compensate the possible alterations in cerebellar cortical activity. References Altman, J., Winfree, A.T., 1977. Postnatal development of the cerebellar cortex in the rat. V. Spatial organization of Purkinje cell perikarya. J. Comp. Neurol. 171, 1 – 16. Barnes, D., Altman, J., 1973. Effects of different schedules of early undernutrition on the preweaning growth of the rat cerebellum. Exp. Neurol. 38, 406 –419. Beas-Za´ rate, C., Del Angel-Meza, A.R., Morales-Villagra´ n, A., Feria-Velasco, A., 1988. Serotonin uptake in the central nervous system of rats fed a corn diet. Comp. Biochem. Physiol. 89C, 173– 177.
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