Reduction of serotonergic neurons in the dorsal raphe due to chronic prenatal administration of a tryptophan-free diet.

Serotonin (5-HT) is a widely studied neurotransmitter which plays an important role in the development and proper functioning of the organism throughout life. The appearance of 5-HT system early in ontogeny suggests the hypothesis that 5-HT plays a regulatory role in neurodevelopment. This study investigated the effect of administration of a tryptophan deficient diet during prenatal development on the morphology and cell population of the dorsal raphe. The experimental diet, containing balanced amounts of carbohydrates, lipids and proteins, was provided to a time-mated group of rats from gestational day 5 until delivery. Control groups were fed with (i) the experimental diet formulation with 0.2% tryptophan added to the mixture, or (ii) a regular chow diet. At delivery, five pups per dam were euthanized. Body and brain weight was measured and brain sections were processed for immunohistochemistry for tryptophan hydroxylase (TrpH) and whole brain 5-HT analysis. Sections containing dorsal raphe were photographed with a light microscope and TrpH positive neurons quantified. Brain weights in the tryptophan deprived group showed no difference as compared with controls while body weights were reduced by 25%. Total numbers of serotonergic neurons at the dorsal raphe in the prenatal tryptophan deficient pups were reduced by 35%. A regional analysis of the dorsal raphe indicated a marked cellular reduction in the medial and caudal sections of the nucleus, which contains the majority of serotonergic neurons, in the tryptophan deprived condition. Quantitative 5-HT analysis showed that the brain concentration was similar among conditions. In conclusion, gestational tryptophan deprivation exerts adverse effects on the development of the 5-HT system, particularly in the dorsal raphe, manifested by decreased numbers of serotonergic neurons as well as altered topography in this important nucleus.

Nutritional tryptophan restriction impairs plasticity of retinotectal axons during the critical period.

The use-dependent specification of neural circuits occurs during post-natal development with a conspicuous influence of environmental factors, such as malnutrition that interferes with the major steps of brain maturation. Serotonin (5-HT), derived exclusively from the essential aminoacid tryptophan, is involved in mechanisms of development and use-dependent plasticity of the central nervous system. We studied the effects of the nutritional restriction of tryptophan in the plasticity of uncrossed retinotectal axons following a retinal lesion to the contralateral retina during the critical period in pigmented rats. Litters were fed through their mothers with a low tryptophan content diet, based on corn and gelatin, a complemented diet with standard tryptophan requirements for rodents or standard laboratory diet. The results suggest a marked reduction in the plasticity of intact axons into denervated territories in the tryptophan restricted group in comparison to control groups. Tryptophan complementation between PND10-21 completely restored retinotectal plasticity. However, the re-introduction of tryptophan after the end of the critical period (between PND28-P41) did not restore the sprouting ability of uncrossed axons suggesting a time-dependent effect to the reversion of plasticity deficits. Tryptophan-restricted animals showed a reduced activity of matrix metalloproteinase-9 and altered expressions of phosphorylated forms of ERK1/2 and AKT. Our results demonstrate the influence of this essential aminoacid as a modulator of neural plasticity during the critical period through the reduction of serotonin content which alters plasticity-related signaling pathways and matrix degradation.

Dietary tryptophan restriction in rats triggers astrocyte cytoskeletal hypertrophy in hippocampus and amygdala.

We have previously reported that dietary tryptophan (TRP) restriction in a rat crucial postnatal developmental stage induces depression-like behavior and alters dendritic spine density in CA1 pyramidal neurons and granule cells of the hippocampus. Due to astrocyte involvement in critical brain mechanisms, it seems worth to investigate possible adaptive changes in the glial population with TRP restriction. Experimental rats were fed with low TRP diet (20% of TRP level of the laboratory rat chow) from postnatal days 30-60. Antibody against glial fibrillary acidic protein (GFAP), a principal intermediate filament in astrocytes, was used to evaluate cytoskeletal hypertrophy and glial proliferation. Our results showed an increase in size and branching of GFAP-immunoreactive (IR) cells in the dorsal hippocampus and amygdala, characteristics of an astrocytic activation. No significant differences were found regarding the number of GFAP-IR cells in both regions. These results indicate that dietary TRP restriction can induce astrocytic activation, hence, provide further evidences supporting the hypothesis that serotonin may also modulate glial morphology.

Nutritional tryptophan restriction and the role of serotonin in development and plasticity of central visual connections.

Tryptophan is an essential amino acid and metabolic precursor of serotonin. Serotonin is both a classical neurotransmitter and a signaling molecule that plays crucial roles in the development of neural circuits and plasticity. The specification of neural circuits in rodents occurs during the postnatal period with conspicuous influence of environmental factors including the nutritional status. Sensory, motor and cognitive systems develop during a critical period, a time window that is crucial to the use-dependent organization of neuronal circuits. This review presents recent experimental findings that disclose some mechanism of tryptophan- and serotonin-dependent plasticity in the developing and adult brain.

Neonatal tryptophan dietary restriction alters development of retinotectal projections in rats.

The specification of sensory neural circuits includes the elimination of transitory axon collaterals/synapses that takes place during early post natal life, an important step for the acquisition of topographical order of sensory systems. Serotonin has been implicated in the patterning of connections in subcortical and cortical circuits. We investigated the effects of the dietary restriction of the only serotonin precursor, tryptophan, on the development of the uncrossed retinotectal pathway in pigmented rats. Litters were fed through their mothers with either a tryptophan restricted, corn and gelatin based diet or a similar control diet complemented with tryptophan during the lactation period. The developmental status of the uncrossed retinotectal terminal fields was studied after the anterograde transport of horseradish peroxidase injected into one eye. We also studied the effects of tryptophan restriction on 5-HT immunoreactivity of raphe neurons, on cAMP levels in the visual layers of the superior colliculus and on protein synthesis among retinal neurons. We found that tryptophan restriction resulted in reduced weight gain among tryptophan restricted rats, without differences in protein synthesis between tryptophan complemented and restricted groups. Tryptophan restriction was also associated with a reduction of serotonin immunoreactive cells in the raphe nuclei and increased cAMP levels in the superior colliculus. Finally we found that neonatal tryptophan restriction resulted in an abnormal patterning of retinotectal topography, which was consistent with a developmental delay in axonal elimination and fine tuning of central connections. These results suggest, therefore, that dietary tryptophan is crucial for the influence of serotonin in the maturation of central visual connections.

Corn feeding during development induces changes in the number of serotonergic neurons in the raphe nuclei.

Serotonin (5-HT) plays a trophic role during brain development; chronic changes in cerebral concentration of this neurotransmitter during the critical stage of development can produce severe damage in the formation of the neural circuits. For the present work a hypoproteic (HYP) diet based on corn (CORN) meal which is deficient in tryptophan (TRY) was given to rats before and during pregnancy, which continued to the offspring until they reached 60 days of age. An isocaloric but hypoproteic diet containing normal amount of TRY, and normal chow (Ch) Purina were given with the same scheme to two groups of rats considered as controls. 5-HT immunohistochemistry was revealed by avidin-biotin complex (ABC) method to quantify serotonergic nerve cells in the nine raphe nuclei. The number of cells immunoreactive to 5-HT immunoreactive (5-HTir) were quantified by means of stereological analysis. Results demonstrated a significant variation in 5-HT expression in the raphe nuclei. Thus, a significant reduction in the number of 5-HTir cells in the rostral raphe nuclei was seen at all ages studied in the animals fed the corn diet, compared to data obtained from the control groups. This decrease was more evident between the postnatal ages of 30 and 60 days. It is concluded that the variations in the available TRY affect the brain cells producing 5-HT and the innervation of their target areas.

Modification of dendritic development.

Since 1890 Ramón y Cajal strongly defended the theory that dendrites and their processes and spines had a function of not just nutrient transport to the cell body, but they had an important conductive role in neural impulse transmission. He extensively discussed and supported this theory in the Volume 1 of his extraordinary book Textura del Sistema Nervioso del Hombre y de los Vertebrados. Also, Don Santiago significantly contributed to a detailed description of the various neural components of the hippocampus and cerebral cortex during development. Extensive investigation has been done in the last Century related to the functional role of these complex brain regions, and their association with learning, memory and some limbic functions. Likewise, the organization and expression of neuropsychological qualities such as memory, exploratory behavior and spatial orientation, among others, depend on the integrity and adequate functional activity of the cerebral cortex and hippocampus. It is known that brain serotonin synthesis and release depend directly and proportionally on the availability of its precursor, tryptophan (TRY). By using a chronic TRY restriction model in rats, we studied their place learning ability in correlation with the dendritic spine density of pyramidal neurons in field CA1 of the hippocampus during postnatal development. We have also reported alterations in the maturation pattern of the ability for spontaneous alternation and task performance evaluating short-term memory, as well as adverse effects on the density of dendritic spines of hippocampal CA1 field pyramidal neurons and on the dendritic arborization and the number of dendritic spines of pyramidal neurons from the third layer of the prefrontal cortex using the same model of TRY restriction. The findings obtained in these studies employing a modified Golgi method, can be interpreted as a trans-synaptic plastic response due to understimulation of serotoninergic receptors located in the hippocampal Ammon's horn and, particularly, on the CA1 field pyramidal neurons, as well as on afferences to the hippocampus which needs to be further investigated.

Cerebral GABA release and GAD activity in protein- and tryptophan-restricted rats during development.

To evaluate the effects on the GABAergic system, Wistar rats were raised on a chronically protein- and tryptophan-restricted diet with 8% protein, based on either Purina chow or corn. There was a significant decrease in both body and cerebral weight in the restricted animals compared with the control group fed with a 23% protein diet. In animals fed mainly corn, glutamic acid decarboxylase (GAD) activity increased significantly at the ages studied (14, 30, and 60 days) in the cerebral cortex and hippocampus. In the same way, gamma-aminobutyric acid (GABA) release decreased significantly in early life in both brain regions, then increased in 30-60-day-old animals corn-fed predominantly in the cerebral cortex. The reduction in GABA release may be attributable to a decrease in GABAergic cell density, which could induce an over-activation of 5-hydroxytryptamine (5-HTergic) receptors, leading in turn to the observed enhancement of GAD activity. Taken together, these results may represent a plastic response by GABAergic neurons to (5-HTergic under-stimulation in mainly corn-fed animals.

A tryptophan-deficient corn-based diet induces plastic responses in cerebellar cortex cells of rat offspring.

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.

Effects of tryptophan depletion on anxiety induced by simulated public speaking

Several lines of evidence point to the participation of serotonin (5HT) in anxiety. Its specific role, however, remains obscure. The objective of the present study was to evaluate the effect of reducing 5HT-neurotransmission through an acute tryptophan depletion on anxiety induced by a simulated public speaking (SPS) test. Two groups of 14-15 subjects were submitted to a 24-h diet with a low or normal content of tryptophan and received an amino acid mixture without (TRY-) or with (TRY+) tryptophan under double-blind conditions. Five hours later they were submitted to the SPS test. The state-trait anxiety inventory (STAI) and the visual analogue mood scale (VAMS) were used to measure subjective anxiety. Both scales showed that SPS induced a significant increase in anxiety. Although no overall difference between groups was found, there was a trend (P = 0.078) to an interaction of group x gender x phases of the SPS, and a separate analysis of each gender showed an increase in anxiety measured by the STAI in females of the TRY- group. The results for the female TRY- group also suggested a greater arousing effect of the SPS test. In conclusion, the tryptophan depletion procedure employed in the present study did not induce a significant general change in subjective anxiety, but tended to induce anxiety in females. This suggests a greater sensitivity of the 5HT system to the effects of the procedure in this gender.

Effects of tryptophan depletion on anxiety induced by simulated public speaking.

Several lines of evidence point to the participation of serotonin (5HT) in anxiety. Its specific role, however, remains obscure. The objective of the present study was to evaluate the effect of reducing 5HT-neurotransmission through an acute tryptophan depletion on anxiety induced by a simulated public speaking (SPS) test. Two groups of 14-15 subjects were submitted to a 24-h diet with a low or normal content of tryptophan and received an amino acid mixture without (TRY-) or with (TRY+) tryptophan under double-blind conditions. Five hours later they were submitted to the SPS test. The state-trait anxiety inventory (STAI) and the visual analogue mood scale (VAMS) were used to measure subjective anxiety. Both scales showed that SPS induced a significant increase in anxiety. Although no overall difference between groups was found, there was a trend (P = 0.078) to an interaction of group x gender x phases of the SPS, and a separate analysis of each gender showed an increase in anxiety measured by the STAI in females of the TRY- group. The results for the female TRY- group also suggested a greater arousing effect of the SPS test. In conclusion, the tryptophan depletion procedure employed in the present study did not induce a significant general change in subjective anxiety, but tended to induce anxiety in females. This suggests a greater sensitivity of the 5HT system to the effects of the procedure in this gender.

Depresión y trastorno de la alimentación / Depression and eating disorders

Los trastornos de la alimentación coexisten frecuentemente con los trastornos depresivos aún cuando la naturaleza de la asociación es todavía controvertida. Las 3 hipótesis fundamentales plantean que los trastornos de la alimentación son pirmarios y la sintomatología depresiva es una consecuencia de ellos, que a la inversa son fenómenos secundarios a un trastorno primario del humor, o bien que ambos tipos de trastornos comparten una multiplicidad de factores que los determinan a ambos. La alta prevalencia de depresión y suicidio en los pacientes con trastornos de la conducta alimentaria, sumando a la alta frecuencia de depresión en las familias de estos pacientes, son una fuerte evidencia de que ambos podrían ser trastornos relacionados. El estudio de los marcadores biológicos ha demostrado una sorprendente similitud entre depresión y trastornos de alimentación. En ambos se ha encontrado evidencia consistente de hipofunción serotoninérgica central, similar respuesta en el test de supresión de dexametasona y efectividad de la luminoterapia tanto para los síntomas depresivos como para los trastornos de la conducta alimentaria. Otro elemento que sugiere una íntima relación entre ambos trastornos es la efectiva respuesta a antidepresivos tricíclicos, inhibidores de la MAO y serotoninérgicos selectivos en el control de los trastornos de la conducta alimentaria que ha sido ampliamente reportada en la literatura. La idea de que la AN y la BN forman parte de un conjunto de trastornos relacionados con hipofunción serotoninérgica parece ser la mejor explicación de los aspectos biológicos involucrados en la génesis de estos trastornos, aspectos que sin embargo deben ser evaluados en el conjunto de factores psicológicos, biográficos y socioculturales que determinan los problemas de salud mental

Chronic tryptophan restriction disrupts grooming chain completion in the rat.

Tryptophan restriction in the diet leads to low levels of brain serotonin. Serotonin has been implicated in the magnitude of novelty-induced grooming behavior, but its possible role in self-grooming chain completion has not been investigated. A tryptophan-deficient diet produced fewer chain-associated face washings, more face washings alone, fewer number of chains, as well as elementary units into chains. Thereafter, an apparent lower threshold for emotional responsiveness also took place. Impairments of the serotoninergic inhibitory activity of striatal and/or nigral dopaminergic terminals is suggested, because corpus striatum has been proposed to be an organizer structure of both the serial ordering as well as in the completion of the self-grooming chains.

The effects of alkali-cooking of corn and supplementation with amaranth seed on its deficiencies in lysine and tryptophan.

Corn, made into tortillas (flat cakes baked from lime-treated corn) is the staple food of Mexico. The amino acid deficiencies of tortillas (TT) and boiled corn (BC), and the supplementary value of amaranth seed (PA), another traditional Mexican food, roasted to the point of "popping", were studied. The feeding tests were 14-day PER trials using weanling rats; each diet contained 8.95% crude protein, all from corn, or with PA providing 3.6% protein and corn the remainder, with vitamin and mineral supplements. In addition each diet was supplemented with lysine (lys) so that tryptophan (trp) would be the first limiting amino acid or vice versa. In no comparison did TT give a significantly different value from BC. With trp limiting, the mean PER for the two corn preparations alone was 1.55, and with PA, 2.22. The amaranth had shown a high trp value (1.55 g/16g N). g N). With lys limiting, the mean PER for corn was 1.15; adding PA failed to improve this. Popping amaranth also reduced its reactive lysine value (by dye-binding) from 5.9 to 4.0 g/16g N. It appears that making tortillas has no adverse effect on protein value, but that 'popping' can reduce the value of amaranth seeds. The higher PER for corn with trp as the limiting amino acid (rather than lys) was due to lower ad libitum food intake with the same weight gain.

Bronchomotor tone in protein-energy malnutrition

The study examined the reactivity of the tracheobronchial tree of rats maintained on low protein and tryptophan-deficient diets. It was found that: (1) Rats maintained on 5 percent protein or triptophan-deficient diets showed little or no weight gain. A 15 percent protein diet was adequate for normal growth of female rats, but not of male rats. (2) Airways of malnourished rats showed significant bronchoconstriction when treated to an acetylcholine (AcCH) concentration of 10 to the 11 power M. The treshold concentration of AcCh for normal rats was 10 to the -5 M. Airways of malnourished rats were also more sensitive to cold. (3) Rehabilitation of the malnourished rats attenuated the response to AcCh. Recovery, however, was not complete. (4) Prior application of phentolamine and atropine markedly reduced the sensitivity of the airways of malnourished rats to AcCh. The results seem to indicate that O-adrenoceptors and the vagus nerve may be involved in the observed increased reactivity of airways of malnourished rats.(AU)

Effect on rats and mice of a diet with low tryptophan content (maize flour).

Female Wistar rats when crowded together become aggressive if fed a diet with low tryptophan (Trp) content (maize, flour, Mf). When isolated during 30 days and fed Mf they show a decrease of weight, of total plasma Trp, and of Trp and 5-hydroxytryptamine (5-HT) in brain stem, but 5-hydroxyindolacetic acid (5-HIAA) of the same cerebral area does not change. These results partially agree with other authors observations. Mice isolated during 47 days and fed with Mf do not show any significant weight variation, the hypermotility of isolated mice on normal diet persists, but 5-HT and 5-HIAA significantly decreases in total brain, an effect that has been observed by another author. The effect on 5-HIAA does not depend on the diet, either balanced or with low Trp content.