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Neither increased nor decreased availability of cortical serotonin (5HT) disturbs barrel field formation in isocaloric undernourished rat pups
Int. J. Devl Neuroscience 20 (2002) 497–501
Neither increased nor decreased availability of cortical serotonin (5HT) disturbs barrel field formation in isocaloric undernourished rat pups Gabriel Gutiérrez-Ospina a,∗ , Gabriel Manjarrez-Gutiérrez b , Cesar González a , Sandra López a , Roc´ıo Herrera b , Ivett Medina-Aguirre a , Jorge Hernández-R c a
Department of Cell Biology and Physiology, Biomedical Research Institute, National Autonomous University of México, México, DF 04510, Mexico b Laboratory of Developmental Neurochemistry, National Medical Center, Mexican Institute of Social Security, México, DF 06703, Mexico c Laboratory of Neurontogeny, Department of Physiology, Biophysics and Neurosciences, Cinvestav, México, DF 07000, Mexico and Faculty of Medicine, University Autonomous of Querétaro, Querétaro 76010, Mexico Received 30 January 2002; received in revised form 20 May 2002; accepted 22 May 2002
Abstract Serotonin (5HT) is expressed transiently in primary sensory areas of the rat neocortex during the establishment of the thalamo-cortical topography and somatotopy. The precise role of 5HT during the specification of neocortical areas is still uncertain. We evaluated the effects of increasing and decreasing cortical serotonin concentrations on the specification of the barrel cortex using a rat model of isocaloric undernutrition. This manipulation increases brain 5HT levels during brain development. Undernourished animals were also treated with p-clorophenylalanine; an inhibitor of 5HT synthesis. Barrels representing the head were readily seen at postnatal day 5 in control and p-clorophenylalanine treated rats. In contrast, undernourished rats treated or not with p-clorophenylalanine showed no barrels representing the head but until postnatal day 7. Chromatographic analyses demonstrated that the concentration of cortical 5HT increased by 50% in undernourished pups during barrel field formation. Control and undernourished animals treated with p-clorophenylalanine had a significant reduction (90%) of 5HT in the cortex. The overall geometry of the barrel field and of individual barrels was similar among animal groups. Our results support that 5HT plays a small role in triggering and timing barrel field somatotopy. © 2002 ISDN. Published by Elsevier Science Ltd. All rights reserved. Keywords: Barrels; Primary somatosensory cortex; Serotonin; Undernutrition; Neurotrophic factors; Developmental timing
1. Introduction In the brain, serotonin (5HT) is produced mainly by neurons located in the raphe nuclei and the reticular region of the lower brainstem. The role of 5HT as a neurotransmitter/modulator is mediated by a number of receptors including the 5HT3 ionotropic receptor, and at least two families of metabotropic receptors couple to G-protein signaling pathways. Besides its involvement in neuronal transmission, 5HT appears to be a developmental signal during embryonic and fetal life (Lauder, 1993). Recent evidence suggests that 5HT is involved also in postnatal brain development. Indeed, a heavy transient serotoninergic innervation penetrates the cerebral cortex as its primary sensory areas are being specified postnatally (D’Amato et al., 1987). The rodent primary somatosensory cortex (S1) has been long used as a model system to study the principles and rules that govern the development and plasticity of the ∗
Corresponding author. Tel.: +52-5-622-3865; fax: +52-5-622-3897. E-mail address: [email protected] (G. Guti´errez-Ospina).
mammalian cortex. Layer IV of S1 contains a body representation formed by cytoarchitectonic modules termed barrels. Each barrel represents groups of mechanosensory receptors located underneath the body surface. Barrels receive a major input via afferents that arise from the ventrobasal complex of the dorsal thalamus. Recent evidence demonstrates that 5HT-containing axons arising from the raphe nuclei reach barrel hollows during the establishment of S1 somatotopy (Bennett-Clarke et al., 1997). Also, developing somatosensory thalamo-cortical axons show 5HT immunoreactivity (Bennett-Clarke et al., 1997), expression of 5HT1B receptors (Bennett-Clarke et al., 1993), and 5HT transporters (Bennett-Clarke et al., 1996; Lebrand et al., 1996) during the formation and initial growth of S1 barrels. Despite circumstantial evidence supporting the participation of 5HT during the early postnatal development of S1, the precise role that this neurotransmitter plays during barrel field formation remains uncertain. Hence, to study further the participation of 5HT on the development of the barrel cortex, we analyzed the formation of barrels in rat pups subjected to isocaloric undernutrition. This manipulation
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increases the concentration of cortical 5HT (Manjarrez et al., 1999), as a physiological response to brain undernutrition. 2. Experimental procedures Control and undernourished rat pups (Wistar, Charles River) were used to carry out the experiments described in the present paper. Isocaloric undernutrition in rats was achieved by following the protocol described in detail previously (Manjarrez et al., 1999). Two subgroups of control and undernourished pups were injected intraperitoneally with p-chlorophenylalanine (PCPA; 300 mg/kg, Sigma–Aldrich) every third day from birth up to the day of sacrifice. Control and experimental animals had free access to feeding and were kept in temperature and light controlled rooms until sacrifice. The number of pups per litter was adjusted to eight animals soon after birth to reduce litter size effects on the animals’ postnatal growth. For histological procedures, control and undernourished rat pups were perfused with normal saline followed by buffered paraformaldehyde (4%). The brains were removed and the cortical mantles dissected and post-fixed in the same fixative for 2 h at room temperature. Cortices were then flattened between two microscope slides and frozen in 2-methyl butane (Riddle et al., 1993). Cortical tangential sections (50 mm) were obtained in a cryostat, mounted on to gelatin-coated slides and stained with cresyl violet. Animals were sacrificed every day, at the same hour, through a period of 5 days beginning at postnatal day (PD) 5 (n = 30 per age). Sections stained with cresyl violet were carefully observed to determine the postnatal day when most barrels of the head representation were formed. An additional set of animals of each group
(n = 6 per age) was used to determine the concentration of 5HT and l-tryptophan (l-Trp) in S1 at PD 2, 4, 6, 8, 10 and 12, by combining the use of high performance liquid chromatography (HPLC) and fluorometric techniques (Manjarrez et al., 1999). Biochemical data were statistically analyzed using Student t-tests. 3. Results 3.1. Body weight in control, undernourished and PCPA-treated rat pups The increase in body weight was equivalent in control and their PCPA-treated counterparts during the first 12 days
Fig. 1. Bar graph illustrating the gain of body weight in control and undernourished rats treated or not with PCPA during the first 11 days of life. Average values (±S.E.M.) of 34 animals per age are depicted. As expected, undernourished pups treated or not with PCPA had significantly lower body weights than their control counterparts. PCPA administration had no appreciable effects on the gain of body weight in control and undernourished rats. Empty bars, control group; dotted bars, control + PCPA group; hatched bars, undernourished group; filled bars, undernourished + PCPA group. Student t-test: () P > 0.05; (∗) P > 0.001.
Fig. 2. Photomicrographs showing tangential sections through layer IV of S1 stained with cresyl violet. A subset of barrels representing the head is illustrated. Control (C) and undernourished (Und) animals of 5 (PD5) and 7 (PD7) days of age, treated or not with PCPA are illustrated. Control and PCPA-treated pups display barrels representing the head from PD5 ahead. In contrast, undernourished rats treated or not with PCPA showed barrels until PD7.
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of life. Undernourished pups treated or not with PCPA had significantly lower body weights at any given age, when compared with control animals. Despite this fact, PCPA-treatment did not affect the gain of body weight in undernourished animals, at least not during the developmental period evaluated in this work (Fig. 1).
concentration of 5HT in S1 was significantly higher at any given day. Repetitive PCPA injections lowered the cortical concentration of 5HT by about 90% in otherwise intact and undernourished animals. Thus, PCPA treatment prevented the increment in 5HT concentrations in S1 of undernourished rats.
3.2. Barrel formation in control, undernourished and PCPA-treated rat pups
4. Discussion
In accord with previous observations, barrels representing the head were present from PN5 ahead in control animals. The administration of PCPA to control animals did not affect the timing of barrel formation. In contrast, barrel formation was delayed by two days in animals subjected to gestational and postnatal isocaloric undernutrition. Such a delay was not affected after treating undernourished rats with PCPA (Fig. 2). 3.3. l-Tryptophan and 5HT concentrations in S1 of control, undernourished and PCPA-treated rats Chromatographic analyses revealed that the concentration of l-Trp in S1 (Fig. 3A) was significantly higher in undernourished rats than in control animals of similar age. Furthermore, in all animal groups studied, there was a decrease of the concentration of l-Trp in S1 with increasing age. The administration of PCPA did not affect the concentration of l-Trp in the developing S1 of treated animals. In contrast to what was seen with l-Trp, 5HT concentrations (Fig. 3B) increased progressively during the first 11 days of life. Such increase was similar in control and undernourished rats, despite the fact that in the latter group the
4.1. Nutritional status, cortical serotonin levels and barrel formation The present results confirm that gestational and postnatal isocaloric undernutrition elevates the concentration of 5HT in the developing cortex of the rat (Manjarrez et al., 1999). This nutritional manipulation associated with a delay of 2 days in the formation of S1 barrels. Although the elevation of the concentration of cortical 5HT might lead to the retardation of barrel formation in rat pups subjected to isocaloric undernutrition, the fact that 5HT depletion after PCPA administration did not shift, prevent or delay further barrel formation in treated control and undernourished animals argues against this possibility. Furthermore, because the gain in body weight was not affected by PCPA, the delay of barrel formation observed in undernourished rats treated with PCPA can not be attributed to an overall impairment of the animal’s growth (Persico et al., 2000). Hence, the delay of barrel formation in animals subjected to dietary restriction appears to be the result of nutritional constraints rather than the result of altered concentrations of cortical 5HT and/or impaired overall body growth. The fact that protein restriction decreases 5HT cortical concentration (Persico et al., 2000) and that barrel formation in animals subjected to protein malnutrition is also delayed (Vondokmai, 1988), lends further support to our conclusion. 4.2. Serotonin as a developmental timing signal in the barrel cortex
Fig. 3. Bar graphs showing the concentration of l-tryptophan (A) and 5HT (B) in S1 of control and undernourished rats treated or not with PCPA during the first 11 days of life. Each age point represents the average value (±S.E.M.) from six experiments made in duplicate. A decrease in l-tryptophan concentration occurred with age in all animal groups. The concentration of 5HT increased in control and undernourished rats during the same time window. Undernourished rats treated or not with PCPA showed significantly higher l-tryptophan concentrations in S1 than their control counterparts at equivalent ages (A). Undernourished rats had increased 5HT levels as compared to age-matched control rats (B). Finally, PCPA administration decreased significantly the concentration of 5HT in S1 of control and undernourished rats. Empty bars, control group; dotted bars, control + PCPA group; hatched bars, undernourished group; filled bars, undernourished + PCPA group. Student t-test: (∗) P > 0.001.
Previously, it has been suggested that 5HT might play a role as a developmental timing signal (Lauder et al., 1980). The delay observed in the formation of cortical layers and barrels in rats treated with 5,7-dihydroxytryptamine, a 5HT depleting neurotoxin, certainly supports this contention (e.g. Osterheld-Haas and Hornung, 1996). In isocaloric undernourished rats, however, the delayed barrel formation associated with an increase in the concentration of cortical 5HT. The depletion of cortical 5HT after PCPA treatment, on the other hand, did not shift the timing of barrel formation in otherwise control and undernourished pups. Our results then undermine the possible role of 5HT as a timing signal during the establishment of S1 somatotopy. In support to this idea, Persico et al. (2000) have shown that most effects (e.g. delayed barrel formation) ascribed to 5HT depleting neurotoxins result from malnutrition, as opposed to changes in the
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concentration of cortical 5HT. Also, Bennett-Clarke et al. (1995) did not report alterations in the timing of barrel formation in rat pups treated with fenfluoramine, an agent that depletes 5HT without causing neurotoxicity. 4.3. Serotonin as a plasticity signal in the barrel cortex The possible participation of 5HT on developmental cortical plasticity have been suggested previously (Osterheld-Haas and Hornung, 1996; Voigt and De Lima, 1991). Indeed, plastic processes along the trigeminal (Rhoades et al., 1990) and visual (Mower, 1991) pathways are thought to be modulated by 5HT. Serotonin has been proposed to regulate the onset (Jonsson and Kasamatsu, 1983) and length (Mower, 1991) of the critical period. The anatomical integrity of the serotoninergic system seems to be required to modify the expression of receptors for other neurotransmitters during cortical plastic responses (Alonso and Soubrie, 1991). Our observations, as well as previous evidence (Vondokmai, 1988; Persico et al., 2000) suggest, however, that 5HT might play a small role, if any, during some types of plasticity. For instance, isocaloric undernutrition and protein malnutrition in rats lead to a generic plastic response characterized by a delay of the formation of cortical barrels. Isocaloric undernutrition (this work) and protein malnutrition (Persico et al., 2000) change, however, the concentration of 5HT in the developing cortex in opposite directions. This suggests that delayed barrel formation in malnourished rats proceeds independent of the levels of cortical 5HT. That 5HT might not be an essential molecule to facilitate plastic responses in the barrel cortex has been also suggested by Turlejski et al. (1997). 4.4. Discrepancies among animals models in serotonin research Cases et al. (1996) and Boylan et al. (2001) showed that the increase in the concentration of cortical 5HT disrupts the geometry of the barrel field, likely by altering the growth of thalamic afferents. In contrast, our results in rats subjected to isocaloric undernutrition indicate that the increment of the concentration of cortical 5HT has no appreciable effects on the overall morphology of the barrel field. This contradiction might be explained by differences in the amount of 5HT available in S1 of MAO-A deficient mice (Cases et al., 1996), in rats treated with clorgyline (Boylan et al., 2001), and in isocaloric undernourished rats (the present study). For instance, while the concentration of 5HT increased 700–900% in monoamine oxidase-A deficient mice, the concentration of 5HT in undernourished rats only raised by about 50%. In addition, Manjarrez et al. (in preparation) has provided evidence showing that an increment in the concentration of cortical 5HT might reduce the expression of 5HT receptors in the developing cortex. Although this negative feedback could decrease the expression of 5HT receptors alike in different animal models, the remaining 5HT receptors
might still be activated in MAO-A deficient mice and clorgyline treated rats because 5HT concentrations reach values as high as 900% (Cases et al., 1996) to 1500% (Boylan et al., 2001) in these animals. This might not be the case for isocaloric undernourished rats whose concentration of cortical 5HT only reach 50%. Finally, another potential source of differences among animal models is that protein synthesis might be particularly depressed, and thus, the growth of the thalamo-cortical afferents greatly impaired, in undernourished rats. 4.5. The role of 5HT during barrel field formation The establishment of topography and somatotopy are two distinct, albeit tightly linked, processes necessary to specify S1 (Dawson and Killackey, 1985). Because barrels display 5HT immunoreactivity right from the onset of their formation and initial growth, it is natural to assume that 5HT might participate in the establishment of S1 somatotopy. The observation that physiological changes in the concentration of cortical 5HT in rats subjected to nutritional deficiencies did not alter barrel field geometry suggest that 5HT plays a minor role in the establishment of S1 somatotopy. This last contention is further substantiated by (1) the lack of effects of PCPA on the barrels’ somatotopy in control and isocaloric undernourished animals (2) the conservation of S1 somatotopy in monoamine oxidase-A deficient mice (Cases et al., 1996), (3) the fact that PCPA treatment in MAO-A deficient mice restores S1 somatotopy instead of disrupting it (Cases et al., 1996) and (4) the observation that clorgyline-treated rats are capable of forming barrels after the critical period of barrel formation, once the treatment is discontinued (Boylan et al., 2001). Although the available evidence supports that 5HT plays a small role during the establishment of S1 somatotopy, we believe that the existing data suggest that 5HT might be more important in fine tuning thalamo-cortical topography by regulating the precision of the growth of thalamic afferents. Indeed, it appears that thalamic afferents in monoamine oxidase-A deficient mice (Cases et al., 1996) and clorgyline treated rats (Boylan et al., 2001) occupy cortical areas (e.g. interbarrel cortex) that normally devoid thalamic connections. Conversely, cortical regions that are normally occupied by thalamic afferents seemed “deprived” of them (i.e. barrels are reduced in size) following non-toxic pharmacological depletion of 5HT (Bennett-Clarke et al., 1995). That 5HT could influence topography by regulating axonal growth should not be surprising because this amine modulates (1) growth cone motility (Haydon et al., 1987) likely by interacting with specific binding sites (Mercado and Hernández, 1992), (2) regulates the expression of extracellular matrix proteins that allow axonal growth (Mayford et al., 1992) and (3) modulates neurite outgrowth in cultured thalamic neurons (Lieske et al., 1999). As for the function of the 5HT innervation present in the already somatotopically organized S1, it is pertinent to recall that 5HT
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promotes glutamatergic neuron differentiation in cortical organotypic cultures (Lavdas et al., 1997). It might then be that the presence of 5HT in cortical barrels facilitates early glutamatergic differentiation of thalamo-cortical projecting neurons and cortical spiny stellate neurons. In addition, 5HT could regulate early synaptogenesis within barrels by stimulating the release of the neurotrophic S-100 protein from astrocytes (Wilson et al., 1998).
Acknowledgements Authors are grateful to Laura Garc´ıa and Patricia Padilla for valuable technical assistance. This work was supported by grants from CONACyT (J28035-N, 30764M, 38615N). Additional funding was provided by Ricardo J. Zevada Foundation and the Mexican Institute of Social Security FP0038/740. References Alonso, R., Soubrie, P., 1991. Effects of serotoninergic denervation on the density and plasticity of brain muscarinic receptors in the rat. Synapse 8, 30–37. Bennett-Clarke, C.A., Leslie, M.J., Chiaia, N.L., Rhoades, R.W., 1993. Serotonin 1B receptors in the developing somatosensory and visual cortices are located on the thalamocortical axons. Proc. Natl. Acad. Sci. U.S.A. 90, 153–157. Bennett-Clarke, C.A., Lane, R.D., Rhoades, R.W., 1995. Fenfluoramine depletes serotonin from the developing cortex and alters thalamocortical organization. Brain Res. 702, 255–260. Bennett-Clarke, C.A., Chiaia, N.L., Rhoades, R.W., 1996. Thalamocortical afferents in rat transiently express high-affinity serotonin uptake sites. Brain Res. 733, 301–306. Bennett-Clarke, C.A., Chaia, N.L., Rhoades, R.W., 1997. Contributions of raphe-cortical and thalamocortical axons to the transient somatotopic pattern of serotonin immunoreactivity in the rat cortex. Somatosen. Mot. Res. 14, 27–33. Boylan, C.B., Kesterson, L.K., Bennett-Clarke, C.A., Chiaia, N.L., Rhoades, R.W., 2001. Neither peripheral nerve input nor cortical NMDA receptor activity are necessary for recovery of a disrupted barrel pattern in rat somatosensory cortex. Dev. Brain Res. 129, 95–106. 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, 297–307. Dawson, D.R., Killackey, H.P., 1985. Distinguishing topography and somatotopy in the thalamocortical proections of the developing rat. Brain Res. 349, 309–313. D’Amato, R.J., Blue, M.E., Largent, B.L., Lynch, D.R., Ledbetter, D.J., Molliver, M.E., Snyder, S.H., 1987. Ontogeny of the serotoninergic projection to rat neocortex: transient expression of a dense innervation to primary sensory areas. Proc. Natl. Acad. Sci. U.S.A. 84, 4322– 4326.
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Haydon, P.G., McCobb, D.P., Kater, S.B., 1987. The regulation of neurite outgrowth, growth cone motility, and electrical synaptogenesis by serotonin. J. Neurobiol. 18, 197–215. Jonsson, G., Kasamatsu, T., 1983. Maturation of monoamine neurotransmitters and receptors in the cat occipital cortex during postnatal critical period. Exp. Brain Res. 50, 449–458. Lauder, J.M., 1993. Neurotransmitters as growth regulatory signals: role of receptors and second messengers. TINS 16, 233–240. Lauder, J.M., Wallace, A.J., Krebs, H., Petrusz, P., 1980. Serotonin as a timing mechanism in neuroembryogenesis. In: Progress in Psychoneuroendocrinology. Elsevier, Amsterdam, pp. 539–556. Lavdas, A.A., Blue, M.E., Lincoln, J., Parnavelas, J.G., 1997. Serotonin promotes the differentiation of glutamate neurons in organotypic slice cultures of the developing cerebral cortex. J. Neurosci. 15, 7872–7880. 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, 823–835. Lieske, V., Bennett-Clarke, C.A., Rhoades, R.W., 1999. Effects of serotonin on neurite outgrowth from thalamic neurons in vitro. Neuroscience 90, 967–974. Manjarrez, G., Magdaleno, V.M., Chagoya, G., Hernández-R, J., 1999. Nutritional recovery does not reverse the activation of brain serotonin synthesis in the ontogenetically malnourished rat. Int. J. Dev. Neurosci. 14, 641–648. Mayford, M., Barzilai, A., Keller, F., Schacher, S., Kandel, E.R., 1992. Modulation of an NCAM-related adhesion molecule with long-term synaptic plasticity in Aplysia. Science 256, 638–644. Mercado, R., Hernández, R.J., 1992. A molecular recognizing system of serotonin in the rat fetal axonal growth cones: uptake and high affinity binding. Dev. Brain Res. 69, 133–137. Mower, G., 1991. Comparison of serotonin 5HT1 receptors and innervation in the visual cortex of normal and dark-reared cats. J. Comp. Neurol. 312, 223–230. Osterheld-Haas, M.-C., Hornung, J.P., 1996. Laminar development of the mouse barrel cortex: effects of neurotoxins against monoamines. Exp. Brain Res. 110, 183–195. Persico, A.M., Altamura, C., Calia, E., Puglisi-Allegra, S., Ventura, R., Luchesse, F., Keller, F., 2000. Serotonin depletion and barrel cortex development: impact of growth impairment versus serotonin effects on thalamocortical endings. Cerebral Cortex 10, 181–191. Riddle, D., Gutiérrez, G., Zheng, D., White, L.E., Richards, A., Purves, D., 1993. Differential metabolic and electrical activity in the somatic sensory of juvenile and adult rats. J. Neurosci. 13, 4193–4213. Rhoades, R.W., Bennett-Clarke, C.A., Chiaia, N.L., White, F.A., Macdonald, G.J., Haring, J.H., Jacquin, M.F., 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, 190–207. Turlejski, K., Djavadian, R.L., Kossut, M., 1997. Neonatal serotonin depletion modifies development but not plasticity in the rat barrel cortex. NeuroReport 8, 1823–1828. Voigt, T., De Lima, A.D., 1991. Serotoninergic innervation of ferret cerebral cortex. II. Postnatal development. J. Comp. Neurol. 314, 415– 428. Vondokmai, R., 1988. Effects of protein malnutrition on development of mouse cortical barrels. J. Comp. Neurol. 191, 283–294. Wilson, C.C., Faber, K.M., Haring, J.H., 1998. Serotonin regulates synaptic connections in the dentate molecular layer of adult rats via 5HT1a receptors: evidence for a glial mechanism. Brain Res. 782, 235–239.
Neither increased nor decreased availability of cortical serotonin (5HT) disturbs barrel field formation in isocaloric undernourished rat pups Gabriel Gutiérrez-Ospina a,∗ , Gabriel Manjarrez-Gutiérrez b , Cesar González a , Sandra López a , Roc´ıo Herrera b , Ivett Medina-Aguirre a , Jorge Hernández-R c a
Department of Cell Biology and Physiology, Biomedical Research Institute, National Autonomous University of México, México, DF 04510, Mexico b Laboratory of Developmental Neurochemistry, National Medical Center, Mexican Institute of Social Security, México, DF 06703, Mexico c Laboratory of Neurontogeny, Department of Physiology, Biophysics and Neurosciences, Cinvestav, México, DF 07000, Mexico and Faculty of Medicine, University Autonomous of Querétaro, Querétaro 76010, Mexico Received 30 January 2002; received in revised form 20 May 2002; accepted 22 May 2002
Abstract Serotonin (5HT) is expressed transiently in primary sensory areas of the rat neocortex during the establishment of the thalamo-cortical topography and somatotopy. The precise role of 5HT during the specification of neocortical areas is still uncertain. We evaluated the effects of increasing and decreasing cortical serotonin concentrations on the specification of the barrel cortex using a rat model of isocaloric undernutrition. This manipulation increases brain 5HT levels during brain development. Undernourished animals were also treated with p-clorophenylalanine; an inhibitor of 5HT synthesis. Barrels representing the head were readily seen at postnatal day 5 in control and p-clorophenylalanine treated rats. In contrast, undernourished rats treated or not with p-clorophenylalanine showed no barrels representing the head but until postnatal day 7. Chromatographic analyses demonstrated that the concentration of cortical 5HT increased by 50% in undernourished pups during barrel field formation. Control and undernourished animals treated with p-clorophenylalanine had a significant reduction (90%) of 5HT in the cortex. The overall geometry of the barrel field and of individual barrels was similar among animal groups. Our results support that 5HT plays a small role in triggering and timing barrel field somatotopy. © 2002 ISDN. Published by Elsevier Science Ltd. All rights reserved. Keywords: Barrels; Primary somatosensory cortex; Serotonin; Undernutrition; Neurotrophic factors; Developmental timing
1. Introduction In the brain, serotonin (5HT) is produced mainly by neurons located in the raphe nuclei and the reticular region of the lower brainstem. The role of 5HT as a neurotransmitter/modulator is mediated by a number of receptors including the 5HT3 ionotropic receptor, and at least two families of metabotropic receptors couple to G-protein signaling pathways. Besides its involvement in neuronal transmission, 5HT appears to be a developmental signal during embryonic and fetal life (Lauder, 1993). Recent evidence suggests that 5HT is involved also in postnatal brain development. Indeed, a heavy transient serotoninergic innervation penetrates the cerebral cortex as its primary sensory areas are being specified postnatally (D’Amato et al., 1987). The rodent primary somatosensory cortex (S1) has been long used as a model system to study the principles and rules that govern the development and plasticity of the ∗
Corresponding author. Tel.: +52-5-622-3865; fax: +52-5-622-3897. E-mail address: [email protected] (G. Guti´errez-Ospina).
mammalian cortex. Layer IV of S1 contains a body representation formed by cytoarchitectonic modules termed barrels. Each barrel represents groups of mechanosensory receptors located underneath the body surface. Barrels receive a major input via afferents that arise from the ventrobasal complex of the dorsal thalamus. Recent evidence demonstrates that 5HT-containing axons arising from the raphe nuclei reach barrel hollows during the establishment of S1 somatotopy (Bennett-Clarke et al., 1997). Also, developing somatosensory thalamo-cortical axons show 5HT immunoreactivity (Bennett-Clarke et al., 1997), expression of 5HT1B receptors (Bennett-Clarke et al., 1993), and 5HT transporters (Bennett-Clarke et al., 1996; Lebrand et al., 1996) during the formation and initial growth of S1 barrels. Despite circumstantial evidence supporting the participation of 5HT during the early postnatal development of S1, the precise role that this neurotransmitter plays during barrel field formation remains uncertain. Hence, to study further the participation of 5HT on the development of the barrel cortex, we analyzed the formation of barrels in rat pups subjected to isocaloric undernutrition. This manipulation
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increases the concentration of cortical 5HT (Manjarrez et al., 1999), as a physiological response to brain undernutrition. 2. Experimental procedures Control and undernourished rat pups (Wistar, Charles River) were used to carry out the experiments described in the present paper. Isocaloric undernutrition in rats was achieved by following the protocol described in detail previously (Manjarrez et al., 1999). Two subgroups of control and undernourished pups were injected intraperitoneally with p-chlorophenylalanine (PCPA; 300 mg/kg, Sigma–Aldrich) every third day from birth up to the day of sacrifice. Control and experimental animals had free access to feeding and were kept in temperature and light controlled rooms until sacrifice. The number of pups per litter was adjusted to eight animals soon after birth to reduce litter size effects on the animals’ postnatal growth. For histological procedures, control and undernourished rat pups were perfused with normal saline followed by buffered paraformaldehyde (4%). The brains were removed and the cortical mantles dissected and post-fixed in the same fixative for 2 h at room temperature. Cortices were then flattened between two microscope slides and frozen in 2-methyl butane (Riddle et al., 1993). Cortical tangential sections (50 mm) were obtained in a cryostat, mounted on to gelatin-coated slides and stained with cresyl violet. Animals were sacrificed every day, at the same hour, through a period of 5 days beginning at postnatal day (PD) 5 (n = 30 per age). Sections stained with cresyl violet were carefully observed to determine the postnatal day when most barrels of the head representation were formed. An additional set of animals of each group
(n = 6 per age) was used to determine the concentration of 5HT and l-tryptophan (l-Trp) in S1 at PD 2, 4, 6, 8, 10 and 12, by combining the use of high performance liquid chromatography (HPLC) and fluorometric techniques (Manjarrez et al., 1999). Biochemical data were statistically analyzed using Student t-tests. 3. Results 3.1. Body weight in control, undernourished and PCPA-treated rat pups The increase in body weight was equivalent in control and their PCPA-treated counterparts during the first 12 days
Fig. 1. Bar graph illustrating the gain of body weight in control and undernourished rats treated or not with PCPA during the first 11 days of life. Average values (±S.E.M.) of 34 animals per age are depicted. As expected, undernourished pups treated or not with PCPA had significantly lower body weights than their control counterparts. PCPA administration had no appreciable effects on the gain of body weight in control and undernourished rats. Empty bars, control group; dotted bars, control + PCPA group; hatched bars, undernourished group; filled bars, undernourished + PCPA group. Student t-test: () P > 0.05; (∗) P > 0.001.
Fig. 2. Photomicrographs showing tangential sections through layer IV of S1 stained with cresyl violet. A subset of barrels representing the head is illustrated. Control (C) and undernourished (Und) animals of 5 (PD5) and 7 (PD7) days of age, treated or not with PCPA are illustrated. Control and PCPA-treated pups display barrels representing the head from PD5 ahead. In contrast, undernourished rats treated or not with PCPA showed barrels until PD7.
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of life. Undernourished pups treated or not with PCPA had significantly lower body weights at any given age, when compared with control animals. Despite this fact, PCPA-treatment did not affect the gain of body weight in undernourished animals, at least not during the developmental period evaluated in this work (Fig. 1).
concentration of 5HT in S1 was significantly higher at any given day. Repetitive PCPA injections lowered the cortical concentration of 5HT by about 90% in otherwise intact and undernourished animals. Thus, PCPA treatment prevented the increment in 5HT concentrations in S1 of undernourished rats.
3.2. Barrel formation in control, undernourished and PCPA-treated rat pups
4. Discussion
In accord with previous observations, barrels representing the head were present from PN5 ahead in control animals. The administration of PCPA to control animals did not affect the timing of barrel formation. In contrast, barrel formation was delayed by two days in animals subjected to gestational and postnatal isocaloric undernutrition. Such a delay was not affected after treating undernourished rats with PCPA (Fig. 2). 3.3. l-Tryptophan and 5HT concentrations in S1 of control, undernourished and PCPA-treated rats Chromatographic analyses revealed that the concentration of l-Trp in S1 (Fig. 3A) was significantly higher in undernourished rats than in control animals of similar age. Furthermore, in all animal groups studied, there was a decrease of the concentration of l-Trp in S1 with increasing age. The administration of PCPA did not affect the concentration of l-Trp in the developing S1 of treated animals. In contrast to what was seen with l-Trp, 5HT concentrations (Fig. 3B) increased progressively during the first 11 days of life. Such increase was similar in control and undernourished rats, despite the fact that in the latter group the
4.1. Nutritional status, cortical serotonin levels and barrel formation The present results confirm that gestational and postnatal isocaloric undernutrition elevates the concentration of 5HT in the developing cortex of the rat (Manjarrez et al., 1999). This nutritional manipulation associated with a delay of 2 days in the formation of S1 barrels. Although the elevation of the concentration of cortical 5HT might lead to the retardation of barrel formation in rat pups subjected to isocaloric undernutrition, the fact that 5HT depletion after PCPA administration did not shift, prevent or delay further barrel formation in treated control and undernourished animals argues against this possibility. Furthermore, because the gain in body weight was not affected by PCPA, the delay of barrel formation observed in undernourished rats treated with PCPA can not be attributed to an overall impairment of the animal’s growth (Persico et al., 2000). Hence, the delay of barrel formation in animals subjected to dietary restriction appears to be the result of nutritional constraints rather than the result of altered concentrations of cortical 5HT and/or impaired overall body growth. The fact that protein restriction decreases 5HT cortical concentration (Persico et al., 2000) and that barrel formation in animals subjected to protein malnutrition is also delayed (Vondokmai, 1988), lends further support to our conclusion. 4.2. Serotonin as a developmental timing signal in the barrel cortex
Fig. 3. Bar graphs showing the concentration of l-tryptophan (A) and 5HT (B) in S1 of control and undernourished rats treated or not with PCPA during the first 11 days of life. Each age point represents the average value (±S.E.M.) from six experiments made in duplicate. A decrease in l-tryptophan concentration occurred with age in all animal groups. The concentration of 5HT increased in control and undernourished rats during the same time window. Undernourished rats treated or not with PCPA showed significantly higher l-tryptophan concentrations in S1 than their control counterparts at equivalent ages (A). Undernourished rats had increased 5HT levels as compared to age-matched control rats (B). Finally, PCPA administration decreased significantly the concentration of 5HT in S1 of control and undernourished rats. Empty bars, control group; dotted bars, control + PCPA group; hatched bars, undernourished group; filled bars, undernourished + PCPA group. Student t-test: (∗) P > 0.001.
Previously, it has been suggested that 5HT might play a role as a developmental timing signal (Lauder et al., 1980). The delay observed in the formation of cortical layers and barrels in rats treated with 5,7-dihydroxytryptamine, a 5HT depleting neurotoxin, certainly supports this contention (e.g. Osterheld-Haas and Hornung, 1996). In isocaloric undernourished rats, however, the delayed barrel formation associated with an increase in the concentration of cortical 5HT. The depletion of cortical 5HT after PCPA treatment, on the other hand, did not shift the timing of barrel formation in otherwise control and undernourished pups. Our results then undermine the possible role of 5HT as a timing signal during the establishment of S1 somatotopy. In support to this idea, Persico et al. (2000) have shown that most effects (e.g. delayed barrel formation) ascribed to 5HT depleting neurotoxins result from malnutrition, as opposed to changes in the
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concentration of cortical 5HT. Also, Bennett-Clarke et al. (1995) did not report alterations in the timing of barrel formation in rat pups treated with fenfluoramine, an agent that depletes 5HT without causing neurotoxicity. 4.3. Serotonin as a plasticity signal in the barrel cortex The possible participation of 5HT on developmental cortical plasticity have been suggested previously (Osterheld-Haas and Hornung, 1996; Voigt and De Lima, 1991). Indeed, plastic processes along the trigeminal (Rhoades et al., 1990) and visual (Mower, 1991) pathways are thought to be modulated by 5HT. Serotonin has been proposed to regulate the onset (Jonsson and Kasamatsu, 1983) and length (Mower, 1991) of the critical period. The anatomical integrity of the serotoninergic system seems to be required to modify the expression of receptors for other neurotransmitters during cortical plastic responses (Alonso and Soubrie, 1991). Our observations, as well as previous evidence (Vondokmai, 1988; Persico et al., 2000) suggest, however, that 5HT might play a small role, if any, during some types of plasticity. For instance, isocaloric undernutrition and protein malnutrition in rats lead to a generic plastic response characterized by a delay of the formation of cortical barrels. Isocaloric undernutrition (this work) and protein malnutrition (Persico et al., 2000) change, however, the concentration of 5HT in the developing cortex in opposite directions. This suggests that delayed barrel formation in malnourished rats proceeds independent of the levels of cortical 5HT. That 5HT might not be an essential molecule to facilitate plastic responses in the barrel cortex has been also suggested by Turlejski et al. (1997). 4.4. Discrepancies among animals models in serotonin research Cases et al. (1996) and Boylan et al. (2001) showed that the increase in the concentration of cortical 5HT disrupts the geometry of the barrel field, likely by altering the growth of thalamic afferents. In contrast, our results in rats subjected to isocaloric undernutrition indicate that the increment of the concentration of cortical 5HT has no appreciable effects on the overall morphology of the barrel field. This contradiction might be explained by differences in the amount of 5HT available in S1 of MAO-A deficient mice (Cases et al., 1996), in rats treated with clorgyline (Boylan et al., 2001), and in isocaloric undernourished rats (the present study). For instance, while the concentration of 5HT increased 700–900% in monoamine oxidase-A deficient mice, the concentration of 5HT in undernourished rats only raised by about 50%. In addition, Manjarrez et al. (in preparation) has provided evidence showing that an increment in the concentration of cortical 5HT might reduce the expression of 5HT receptors in the developing cortex. Although this negative feedback could decrease the expression of 5HT receptors alike in different animal models, the remaining 5HT receptors
might still be activated in MAO-A deficient mice and clorgyline treated rats because 5HT concentrations reach values as high as 900% (Cases et al., 1996) to 1500% (Boylan et al., 2001) in these animals. This might not be the case for isocaloric undernourished rats whose concentration of cortical 5HT only reach 50%. Finally, another potential source of differences among animal models is that protein synthesis might be particularly depressed, and thus, the growth of the thalamo-cortical afferents greatly impaired, in undernourished rats. 4.5. The role of 5HT during barrel field formation The establishment of topography and somatotopy are two distinct, albeit tightly linked, processes necessary to specify S1 (Dawson and Killackey, 1985). Because barrels display 5HT immunoreactivity right from the onset of their formation and initial growth, it is natural to assume that 5HT might participate in the establishment of S1 somatotopy. The observation that physiological changes in the concentration of cortical 5HT in rats subjected to nutritional deficiencies did not alter barrel field geometry suggest that 5HT plays a minor role in the establishment of S1 somatotopy. This last contention is further substantiated by (1) the lack of effects of PCPA on the barrels’ somatotopy in control and isocaloric undernourished animals (2) the conservation of S1 somatotopy in monoamine oxidase-A deficient mice (Cases et al., 1996), (3) the fact that PCPA treatment in MAO-A deficient mice restores S1 somatotopy instead of disrupting it (Cases et al., 1996) and (4) the observation that clorgyline-treated rats are capable of forming barrels after the critical period of barrel formation, once the treatment is discontinued (Boylan et al., 2001). Although the available evidence supports that 5HT plays a small role during the establishment of S1 somatotopy, we believe that the existing data suggest that 5HT might be more important in fine tuning thalamo-cortical topography by regulating the precision of the growth of thalamic afferents. Indeed, it appears that thalamic afferents in monoamine oxidase-A deficient mice (Cases et al., 1996) and clorgyline treated rats (Boylan et al., 2001) occupy cortical areas (e.g. interbarrel cortex) that normally devoid thalamic connections. Conversely, cortical regions that are normally occupied by thalamic afferents seemed “deprived” of them (i.e. barrels are reduced in size) following non-toxic pharmacological depletion of 5HT (Bennett-Clarke et al., 1995). That 5HT could influence topography by regulating axonal growth should not be surprising because this amine modulates (1) growth cone motility (Haydon et al., 1987) likely by interacting with specific binding sites (Mercado and Hernández, 1992), (2) regulates the expression of extracellular matrix proteins that allow axonal growth (Mayford et al., 1992) and (3) modulates neurite outgrowth in cultured thalamic neurons (Lieske et al., 1999). As for the function of the 5HT innervation present in the already somatotopically organized S1, it is pertinent to recall that 5HT
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promotes glutamatergic neuron differentiation in cortical organotypic cultures (Lavdas et al., 1997). It might then be that the presence of 5HT in cortical barrels facilitates early glutamatergic differentiation of thalamo-cortical projecting neurons and cortical spiny stellate neurons. In addition, 5HT could regulate early synaptogenesis within barrels by stimulating the release of the neurotrophic S-100 protein from astrocytes (Wilson et al., 1998).
Acknowledgements Authors are grateful to Laura Garc´ıa and Patricia Padilla for valuable technical assistance. This work was supported by grants from CONACyT (J28035-N, 30764M, 38615N). Additional funding was provided by Ricardo J. Zevada Foundation and the Mexican Institute of Social Security FP0038/740. References Alonso, R., Soubrie, P., 1991. Effects of serotoninergic denervation on the density and plasticity of brain muscarinic receptors in the rat. Synapse 8, 30–37. Bennett-Clarke, C.A., Leslie, M.J., Chiaia, N.L., Rhoades, R.W., 1993. Serotonin 1B receptors in the developing somatosensory and visual cortices are located on the thalamocortical axons. Proc. Natl. Acad. Sci. U.S.A. 90, 153–157. Bennett-Clarke, C.A., Lane, R.D., Rhoades, R.W., 1995. Fenfluoramine depletes serotonin from the developing cortex and alters thalamocortical organization. Brain Res. 702, 255–260. Bennett-Clarke, C.A., Chiaia, N.L., Rhoades, R.W., 1996. Thalamocortical afferents in rat transiently express high-affinity serotonin uptake sites. Brain Res. 733, 301–306. Bennett-Clarke, C.A., Chaia, N.L., Rhoades, R.W., 1997. Contributions of raphe-cortical and thalamocortical axons to the transient somatotopic pattern of serotonin immunoreactivity in the rat cortex. Somatosen. Mot. Res. 14, 27–33. Boylan, C.B., Kesterson, L.K., Bennett-Clarke, C.A., Chiaia, N.L., Rhoades, R.W., 2001. Neither peripheral nerve input nor cortical NMDA receptor activity are necessary for recovery of a disrupted barrel pattern in rat somatosensory cortex. Dev. Brain Res. 129, 95–106. 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, 297–307. Dawson, D.R., Killackey, H.P., 1985. Distinguishing topography and somatotopy in the thalamocortical proections of the developing rat. Brain Res. 349, 309–313. D’Amato, R.J., Blue, M.E., Largent, B.L., Lynch, D.R., Ledbetter, D.J., Molliver, M.E., Snyder, S.H., 1987. Ontogeny of the serotoninergic projection to rat neocortex: transient expression of a dense innervation to primary sensory areas. Proc. Natl. Acad. Sci. U.S.A. 84, 4322– 4326.
501
Haydon, P.G., McCobb, D.P., Kater, S.B., 1987. The regulation of neurite outgrowth, growth cone motility, and electrical synaptogenesis by serotonin. J. Neurobiol. 18, 197–215. Jonsson, G., Kasamatsu, T., 1983. Maturation of monoamine neurotransmitters and receptors in the cat occipital cortex during postnatal critical period. Exp. Brain Res. 50, 449–458. Lauder, J.M., 1993. Neurotransmitters as growth regulatory signals: role of receptors and second messengers. TINS 16, 233–240. Lauder, J.M., Wallace, A.J., Krebs, H., Petrusz, P., 1980. Serotonin as a timing mechanism in neuroembryogenesis. In: Progress in Psychoneuroendocrinology. Elsevier, Amsterdam, pp. 539–556. Lavdas, A.A., Blue, M.E., Lincoln, J., Parnavelas, J.G., 1997. Serotonin promotes the differentiation of glutamate neurons in organotypic slice cultures of the developing cerebral cortex. J. Neurosci. 15, 7872–7880. 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, 823–835. Lieske, V., Bennett-Clarke, C.A., Rhoades, R.W., 1999. Effects of serotonin on neurite outgrowth from thalamic neurons in vitro. Neuroscience 90, 967–974. Manjarrez, G., Magdaleno, V.M., Chagoya, G., Hernández-R, J., 1999. Nutritional recovery does not reverse the activation of brain serotonin synthesis in the ontogenetically malnourished rat. Int. J. Dev. Neurosci. 14, 641–648. Mayford, M., Barzilai, A., Keller, F., Schacher, S., Kandel, E.R., 1992. Modulation of an NCAM-related adhesion molecule with long-term synaptic plasticity in Aplysia. Science 256, 638–644. Mercado, R., Hernández, R.J., 1992. A molecular recognizing system of serotonin in the rat fetal axonal growth cones: uptake and high affinity binding. Dev. Brain Res. 69, 133–137. Mower, G., 1991. Comparison of serotonin 5HT1 receptors and innervation in the visual cortex of normal and dark-reared cats. J. Comp. Neurol. 312, 223–230. Osterheld-Haas, M.-C., Hornung, J.P., 1996. Laminar development of the mouse barrel cortex: effects of neurotoxins against monoamines. Exp. Brain Res. 110, 183–195. Persico, A.M., Altamura, C., Calia, E., Puglisi-Allegra, S., Ventura, R., Luchesse, F., Keller, F., 2000. Serotonin depletion and barrel cortex development: impact of growth impairment versus serotonin effects on thalamocortical endings. Cerebral Cortex 10, 181–191. Riddle, D., Gutiérrez, G., Zheng, D., White, L.E., Richards, A., Purves, D., 1993. Differential metabolic and electrical activity in the somatic sensory of juvenile and adult rats. J. Neurosci. 13, 4193–4213. Rhoades, R.W., Bennett-Clarke, C.A., Chiaia, N.L., White, F.A., Macdonald, G.J., Haring, J.H., Jacquin, M.F., 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, 190–207. Turlejski, K., Djavadian, R.L., Kossut, M., 1997. Neonatal serotonin depletion modifies development but not plasticity in the rat barrel cortex. NeuroReport 8, 1823–1828. Voigt, T., De Lima, A.D., 1991. Serotoninergic innervation of ferret cerebral cortex. II. Postnatal development. J. Comp. Neurol. 314, 415– 428. Vondokmai, R., 1988. Effects of protein malnutrition on development of mouse cortical barrels. J. Comp. Neurol. 191, 283–294. Wilson, C.C., Faber, K.M., Haring, J.H., 1998. Serotonin regulates synaptic connections in the dentate molecular layer of adult rats via 5HT1a receptors: evidence for a glial mechanism. Brain Res. 782, 235–239.