Improvement of neuronal differentiation by carbon monoxide: Role of pentose phosphate pathway.

Over the last decades, the silent-killer carbon monoxide (CO) has been shown to also be an endogenous cytoprotective molecule able to inhibit cell death and modulate mitochondrial metabolism. Neuronal metabolism is mostly oxidative and neurons also use glucose for maintaining their anti-oxidant status by generation of reduced glutathione (GSH) via the pentose-phosphate pathway (PPP). It is established that neuronal differentiation depends on reactive oxygen species (ROS) generation and signalling, however there is a lack of information about modulation of the PPP during adult neurogenesis. Thus, the main goal of this study was to unravel the role of CO on cell metabolism during neuronal differentiation, particularly by targeting PPP flux and GSH levels as anti-oxidant system. A human neuroblastoma SH-S5Y5 cell line was used, which differentiates into post-mitotic neurons by treatment with retinoic acid (RA), supplemented or not with CO-releasing molecule-A1 (CORM-A1). SH-SY5Y cell differentiation supplemented with CORM-A1 prompted an increase in neuronal yield production. It did, however, not alter glycolytic metabolism, but increased the PPP. In fact, CORM-A1 treatment stimulated (i) mRNA expression of 6-phosphogluconate dehydrogenase (PGDH) and transketolase (TKT), which are enzymes for oxidative and non-oxidative phases of the PPP, respectively and (ii) protein expression and activity of glucose 6-phosphate dehydrogenase (G6PD) the rate-limiting enzyme of the PPP. Likewise, whenever G6PD was knocked-down CO-induced improvement on neuronal differentiation was reverted, while pharmacological inhibition of GSH synthesis did not change CO's effect on the improvement of neuronal differentiation. Both results indicate the key role of PPP in CO-modulation of neuronal differentiation. Furthermore, at the end of SH-SY5Y neuronal differentiation process, CORM-A1 supplementation increased the ratio of reduced and oxidized glutathione (GSH/GSSG) without alteration of GSH metabolism. These data corroborate with PPP stimulation. In conclusion, CO improves neuronal differentiation of SH-S5Y5 cells by stimulating the PPP and modulating the GSH system.

Carbon monoxide improves neuronal differentiation and yield by increasing the functioning and number of mitochondria.

The process of cell differentiation goes hand-in-hand with metabolic adaptations, which are needed to provide energy and new metabolites. Carbon monoxide (CO) is an endogenous cytoprotective molecule able to inhibit cell death and improve mitochondrial metabolism. Neuronal differentiation processes were studied using the NT2 cell line, which is derived from human testicular embryonic teratocarcinoma and differentiates into post-mitotic neurons upon retinoic acid treatment. CO-releasing molecule A1 (CORM-A1) was used do deliver CO into cell culture. CO treatment improved NT2 neuronal differentiation and yield, since there were more neurons and the total cell number increased following the differentiation process. CO supplementation enhanced the mitochondrial population in post-mitotic neurons derived from NT2 cells, as indicated by an increase in mitochondrial DNA. CO treatment during neuronal differentiation increased the extent of the classical metabolic change that occurs during neuronal differentiation, from glycolytic to more oxidative metabolism, by decreasing the ratio of lactate production and glucose consumption. The expression of pyruvate and lactate dehydrogenases was higher, indicating an augmented oxidative metabolism. Moreover, these findings were corroborated by an increased percentage of (13) C incorporation from [U-(13) C]glucose into the tricarboxylic acid cycle metabolites malate and citrate, and also glutamate and aspartate in CO-treated cells. Finally, under low levels of oxygen (5%), which enhances glycolytic metabolism, some of the enhancing effects of CO on mitochondria were not observed. In conclusion, our data show that CO improves neuronal and mitochondrial yield by stimulation of tricarboxylic acid cycle activity, and thus oxidative metabolism of NT2 cells during the process of neuronal differentiation. The process of cell differentiation is coupled with metabolic adaptations. Carbon monoxide (CO) is an endogenous cytoprotective gasotransmitter able to prevent cell death and improve mitochondrial metabolism. Herein CO supplementation improved neuronal differentiation yield, by enhancing mitochondrial population and promoting the classical metabolic change that occurs during neuronal differentiation, from glycolytic to oxidative metabolism.

Functional and phenotypic differences of pure populations of stem cell-derived astrocytes and neuronal precursor cells.

Availability of homogeneous astrocyte populations would facilitate research concerning cell plasticity (metabolic and transcriptional adaptations; innate immune responses) and cell cycle reactivation. Current protocols to prepare astrocyte cultures differ in their final content of immature precursor cells, preactivated cells or entirely different cell types. A new method taking care of all these issues would improve research on astrocyte functions. We found here that the exposure of a defined population of pluripotent stem cell-derived neural stem cells (NSC) to BMP4 results in pure, nonproliferating astrocyte cultures within 24-48 h. These murine astrocytes generated from embryonic stem cells (mAGES) expressed the positive markers GFAP, aquaporin 4 and GLT-1, supported neuronal function, and acquired innate immune functions such as the response to tumor necrosis factor and interleukin 1. The protocol was applicable to several normal or disease-prone pluripotent cell lines, and the corresponding mAGES all exited the cell cycle and lost most of their nestin expression, in contrast to astrocytes generated by serum-addition or obtained as primary cultures. Comparative gene expression analysis of mAGES and NSC allowed quantification of differences between the two cell types and a definition of an improved marker set to define astrocytes. Inclusion of several published data sets in this transcriptome comparison revealed the similarity of mAGES with cortical astrocytes in vivo. Metabolic analysis of homogeneous NSC and astrocyte populations revealed distinct neurochemical features: both cell types synthesized glutamine and citrate, but only mature astrocytes released these metabolites. Thus, the homogeneous cultures allowed an improved definition of NSC and astrocyte features.

Dietary supplementation with acetyl-l-carnitine in seizure treatment of pentylenetetrazole kindled mice.

In spite of the availability of new antiepileptic drugs a considerable number of epilepsy patients still have pharmacoresistant seizures, and thus there is a need for novel approaches. Acetyl-l-carnitine (ALCAR), which delivers acetyl units to mitochondria for acetyl-CoA production, has been shown to improve brain energy homeostasis and protects against various neurotoxic insults. To our knowledge, this is the first study of ALCAR's effect on metabolism in pentylenetetrazole (PTZ) kindled mice. ALCAR or the commonly used antiepileptic drug valproate, was added to the drinking water of mice for 25days, and animals were injected with PTZ or saline three times a week during the last 21 days. In order to investigate ALCAR's effects on glucose metabolism, mice were injected with [1-(13)C]glucose 15 min prior to microwave fixation. Brain extracts from cortex and the hippocampal formation (HF) were studied using (1)H and (13)C NMR spectroscopy and HPLC. PTZ kindling caused glucose hypometabolism, evidenced by a reduction in both glycolysis and TCA cycle turnover in both brain regions investigated. Glutamatergic and GABAergic neurons were affected in cortex and HF, but the amount of glutamate was only reduced in HF. Slight astrocytic involvement could be detected in the cortex. Interestingly, the dopamine content was increased in the HF. ALCAR attenuated the PTZ induced reduction in [3-(13)C]alanine and the increase in dopamine in the HF. However, TCA cycle metabolism was not different from that seen in PTZ kindled animals. In conclusion, even though ALCAR did not delay the kindling process, it did show some promising ameliorative effects, worthy of further investigation.

Chronic acetyl-L-carnitine alters brain energy metabolism and increases noradrenaline and serotonin content in healthy mice.

Acetyl-L-carnitine (ALCAR), the short-chain ester of carnitine, is a common dietary supplement readily available in health food stores, claimed to improve energy levels and muscle strength. ALCAR has numerous effects on brain and muscle metabolism, protects against neurotoxic insults and may be an effective treatment for certain forms of depression. However, little is known about the effect of chronic ALCAR supplementation on the brain metabolism of healthy mice. Here, we investigated ALCAR's effect on cerebral energy and neurotransmitter metabolism after supplementing the drinking water of mice with ALCAR for 25 days, providing a daily dose of about 0.5 g/kg. Thereafter the animals were injected with [1-(13)C]glucose, and (13)C incorporation into and levels of various metabolites were quantified in extracts of the hippocampal formation (HF) and cortex using (1)H- and (13)C-nuclear magnetic resonance (NMR) spectroscopy and high performance liquid chromatography (HPLC). Increased glucose levels were detected in both regions together with a decreased amount of [3-(13)C]lactate, but no alterations in incorporation of (13)C derived from [1-(13)C]glucose into the amino acids glutamate, GABA and glutamine. These findings are consistent with decreased metabolism of glucose to lactate but not via the TCA cycle. Higher amounts of the sum of adenosine nucleotides, phosphocreatine and the phosphocreatine/creatine ratio found in the cortex of ALCAR-treated mice are indicative of increased energy levels. Furthermore, ALCAR supplementation increased the levels of the neurotransmitters noradrenaline in the HF and serotonin in cortex, consistent with ALCAR's potential efficacy for depressive symptoms. Other ALCAR-induced changes observed included reduced amounts of GABA in the HF and increased myo-inositol. In conclusion, chronic ALCAR supplementation decreased glucose metabolism to lactate, resulted in increased energy metabolite and altered monoamine neurotransmitter levels in the mouse brain.

ß-Hydroxybutyrate is the preferred substrate for GABA and glutamate synthesis while glucose is indispensable during depolarization in cultured GABAergic neurons.

The ketogenic diet has multiple beneficial effects not only in treatment of epilepsy, but also in that of glucose transporter 1 deficiency, cancer, Parkinson's disease, obesity and pain. Thus, there is an increasing interest in understanding the mechanism behind this metabolic therapy. Patients on a ketogenic diet reach high plasma levels of ketone bodies, which are used by the brain as energy substrates. The interaction between glucose and ketone bodies is complex and there is still controversy as to what extent it affects the homeostasis of the neurotransmitters glutamate, aspartate and GABA. The present study was conducted to study this metabolic interaction in cultured GABAergic neurons exposed to different combinations of (13)C-labeled and unlabeled glucose and ß-hydroxybutyrate. Depolarization was induced and the incorporation of (13)C into glutamate, GABA and aspartate was analyzed. The presence of ß-hydroxybutyrate together with glucose did not affect the total GABA content but did, however, decrease the aspartate content to a lower value than when either glucose or ß-hydroxybutyrate was employed alone. When combinations of the two substrates were used (13)C-atoms from ß-hydroxybutyrate were found in all three amino acids to a greater extent than (13)C-atoms from glucose, but only the (13)C contribution from [1,6-(13)C]glucose increased upon depolarization. In conclusion, ß-hydroxybutyrate was preferred over glucose as substrate for amino acid synthesis but the total content of aspartate decreased when both substrates were present. Furthermore only the use of glucose increased upon depolarization.

[2,4-(13)C]ß-hydroxybutyrate metabolism in astrocytes and C6 glioblastoma cells.

This study was undertaken to determine if the ketogenic diet could be useful for glioblastoma patients. The hypothesis tested was whether glioblastoma cells can metabolize ketone bodies. Cerebellar astrocytes and C6 glioblastoma cells were incubated in glutamine and serum free medium containing [2,4-(13)C]ß-hydroxybutyrate (BHB) with and without glucose. Furthermore, C6 cells were incubated with [1-(13)C]glucose in the presence and absence of BHB. Cell extracts were analyzed by mass spectrometry and media by (1)H magnetic resonance spectroscopy and HPLC. Using [2,4-(13)C]BHB and [1-(13)C]glucose it could be shown that C6 cells, in analogy to astrocytes, had efficient mitochondrial activity, evidenced by (13)C labeling of glutamate, glutamine and aspartate. However, in the presence of glucose, astrocytes were able to produce and release glutamine, whereas this was not accomplished by the C6 cells, suggesting lack of anaplerosis in the latter. We hypothesize that glioblastoma cells kill neurons by not supplying the necessary glutamine, and by releasing glutamate.

The role of glia in neuronal recovery following anoxia: In vitro evidence of neuronal adaptation.

We investigated the effects of 3h of anoxia on metabolism of neurons and astrocytes, using a robust cell-based model system that mimics closely the living tissue milieu, i.e., in 3D neural aggregates cultured in bioreactors. Cells were incubated simultaneously with [1-(13)C]glucose and [1,2-(13)C]acetate; and, the gliotoxin fluorocitrate (FC) was used for glial tricarboxylic acid (TCA) cycle inhibition to assess the role of astrocytes for neuronal metabolism after oxygen deprivation. Results show that culture viability was not compromised by exposure to anoxia with and without FC. Interaction between astrocytes and glutamatergic neurons was altered due to anoxia: labeling in glutamine from [1-(13)C]glucose was decreased, whereas that in glutamate from [1,2-(13)C]acetate was increased. In contrast, GABA labeling was not affected by anoxia. It was shown that anoxia did not affect astrocytic capacity to synthesize glutamine in the reoxygenation period. The selective action of FC on astrocytes was confirmed. However, the presence of small amounts of glutamate and GABA labeled from acetate indicated residual activity of the glial TCA cycle. Although major metabolic changes were found due to FC-treatment, the intracellular pool of GABA was kept unchanged. Overall, our data clearly confirm that the glutamate-glutamine cycle depends on astrocytic TCA cycle activity and that mitochondrial impairment of astrocytes will ultimately stop metabolic trafficking between astrocytes and glutamatergic neurons. Additionally, our data suggest a metabolic independence of GABAergic neurons from astrocytes even after situations of complete oxygen depletion.

Mild reduction in the activity of the alpha-ketoglutarate dehydrogenase complex elevates GABA shunt and glycolysis.

Diminished energy metabolism and reduced activity of brain alpha-ketoglutarate dehydrogenase complex (KGDHC) occur in a number of neurodegenerative diseases. The relation between diminished KGDHC activity and altered energy metabolism is unknown. The present study tested whether a reduction in the KGDHC activity would alter cellular metabolism by comparing metabolism of [U-13C]glucose in a human embryonic kidney cell line (E2k100) to one in which the KGDHC activity was about 70% of control (E2k67). After a 2 h incubation of the cells with [U-13C]glucose, the E2k67 cells showed a greater increase in 13C labeling of alanine compared with the E2k100 cells. This suggested an increase in glycolysis. Furthermore, an increase in labeled lactate after 12 h incubation supported the suggestion of an increased glycolysis in the E2k67 cells. Increased GABA shunt in the E2k67 cells was indicated by increased 13C labeling of GABA at both 2 and 12 h compared with the control cells. GABA concentration as determined by HPLC was also increased in the E2k67 cells compared with the control cells. However, the GABA shunt was not sufficient to normalize metabolism in the E2k67 cells compared with control at 2 or 12 h. However, by 24 h metabolism had normalized (i.e. labeling was similar in E2k67 and E2k100). Thus, the data are consistent with an enhanced glycolysis and GABA shunt in response to a mild reduction in KGDHC. Our findings indicate that a mild change in KGDHC activity can lead to large changes in metabolism. The changes may maintain normal energy metabolism but make the cells more vulnerable to perturbations such as occur with oxidants.

Characterization of brain metastases using high-resolution magic angle spinning MRS.

The objectives of this study were to (a) explore the spectral characteristics of brain metastases, focusing on the origin of the primary cancer, and (b) evaluate the correlation with clinical outcome using multivariate analysis. High-resolution magic angle spinning (HR-MAS) MR spectra (n = 26) were obtained from 16 patients with brain metastases using a Bruker Avance DRX600 instrument. Standard pulse-acquired and spin-echo (TE 32 and 285 ms) (1)H spectra were obtained. These were examined using principal component analysis (PCA) and partial least squares regression analysis (PLS) relating spectral data to clinical outcome. The PCA score plot of pulse-acquired HR-MAS spectra showed a trend of clustering due to the origin of the metastases, mainly based on differences in the lipid signals at 1.3 and 0.9 ppm. With PLS, spectra of patients who died less than 5 months after surgery appeared to cluster in the lower left quadrant of the score plot. These preliminary results on brain metastasis classification and prediction of survival must be validated in a larger patient cohort. However, the possibility of differentiating metastases according to origin and predicting survival on the basis of HR-MAS spectra suggests that this method may be useful for diagnosing and planning treatment for brain metastases and also for guiding decisions about terminating further treatment.

Metabolic profiling of human brain metastases using in vivo proton MR spectroscopy at 3T.

BACKGROUND: Metastases to the central nervous system from different primary cancers are an oncologic challenge as the overall prognosis for these patients is generally poor. The incidence of brain metastases varies with type of primary cancer and is probably increasing due to improved therapies of extracranial metastases prolonging patient's overall survival and thereby time for brain metastases to develop. In addition, the greater access to improved neuroimaging techniques can provide earlier diagnosis. The aim of this study was to investigate the feasibility of using proton magnetic resonance spectroscopy (MRS) and multivariate analyses to characterize brain metastases originating from different primary cancers, to assess changes in spectra during radiation treatment and to correlate the spectra to clinical outcome after treatment. METHODS: Patients (n = 26) with brain metastases were examined using single voxel MRS at a 3T clinical MR system. Five patients were excluded due to poor spectral quality. The spectra were obtained before start (n = 21 patients), immediately after (n = 6 patients) and two months after end of treatment (n = 4 patients). Principal component analysis (PCA) and partial least square regression analysis (PLS) were applied in order to identify clustering of spectra due to origin of metastases and to relate clinical outcome (survival) of the patients to spectral data from the first MR examination. RESULTS: The PCA results indicated that brain metastases from primary lung and breast cancer were separated into two clusters, while the metastases from malignant melanomas showed no uniformity. The PLS analysis showed a significant correlation between MR spectral data and survival five months after MRS before start of treatment. CONCLUSION: MRS determined metabolic profiles analysed by PCA and PLS might give valuable clinical information when planning and evaluating the treatment of brain metastases, and also when deciding to terminate further therapies.

Impaired glutamine metabolism in NMDA receptor hypofunction induced by MK801.

Paradoxically, glutamate receptor antagonists have neurotoxic and psychotogenic properties in addition to their neuroprotective potential during excessive glutamate release. In the present study the non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist MK801 was used to examine glial-neuronal interactions in NMDA receptor hypofunction. Rats were given a subanesthetic dose of MK801 together with [1-13C]glucose and [1,2-13C]acetate, and brains were removed 20 min later. Analyses of extracts from cingulate, retrosplenial plus middle frontal cortices (CRFC) and temporal lobe were performed using HPLC and 13C and 1H nuclear magnetic resonance spectroscopy. Hypofunction of the NMDA receptor induced similar changes in both brain areas investigated; however, the changes were most pronounced in the temporal lobe. Generally, only labeling from [1-13C]glucose was affected by MK801. In CRFC and temporal lobe amounts of both labeled and unlabeled glutamine were increased, whereas those of aspartate were decreased. In the CRFC the decrease in labeling of aspartate was greater than the decrease in concentration, leading to decreased 13C enrichment. In temporal lobe, not in CRFC, increased concentrations of glutamate, GABA, succinate, glutathione and inositol were detected together with increased labeling of GABA and succinate from [1-13C]glucose. 13C Enrichment was decreased in glutamate and increased in succinate. The results point towards a disturbance in glutamate-glutamine cycling and thus interaction between neurons and glia, since labeling of glutamate and glutamine from glucose was affected differently.

Effects of methylmercury on primary brain cells in mono- and co-culture.

We report on the uptake of MeHg in astrocytes and neurons, as well as specific indicators of neurotoxicity. Cerebellar granule neurons and astrocytes separately and in co-culture were cultured in the presence of MeHg and changes in 3-[4, 5-dimethylthiazol-2-yl]-2, 5 diphenyltetrazolium bromide (MTT)-reduction, lactate dehydrogenase (LDH) leakage, and cellular content of glutathione and amino acids were used as indicators of MeHg toxicity. Mitochondria in cortical astrocytes were slightly more sensitive than those in cerebellar astrocytes to the toxic effects of MeHg; furthermore, cellular integrity was better preserved in cerebellar astrocytes. When neurons and astrocytes from cerebellum were incubated in separable co-cultures using inserts, the astrocytes showed cellular damage at lower exposure to MeHg while neurons showed less changes compared to respective cell types in mono-cultures. Mercury uptake studies at 25 microM MeHg (10% serum present) showed that for neurons in co-culture the uptake was 1/3 compared to mono-cultures. In contrast, for astrocytes in co-culture, uptake was increased by 75%. A MeHg concentration-dependent increase of glutamate content in mono-cultures was noted. When MeHg concentration was increased to 10, 25, or 50 microM, neurons in co-cultures decreased their glutamate content, whereas astrocytes showed an increase. Other amino acids, such as glutamine, serine, valine, isoleucine, taurine, and phenylalanine were unaffected by MeHg. Glutathione content showed MeHg concentration-dependent changes in astrocytes and was increased in neurons in co-culture incubated with 5 microM MeHg. In conclusion, astrocytes appear to increase neuronal resistance, indicating a possible protective role for astrocytes in MeHg neurotoxicity.

Metabolism is normal in astrocytes in chronically epileptic rats: a (13)C NMR study of neuronal-glial interactions in a model of temporal lobe epilepsy.

The aim of the present work was to study potential disturbances in metabolism and interactions between neurons and glia in the lithium-pilocarpine model of temporal lobe epilepsy. Rats chronically epileptic for 1 month received [1-(13)C]glucose, a substrate for neurons and astrocytes, and [1,2-(13)C]acetate, a substrate for astrocytes only. Analyses of extracts from cerebral cortex, cerebellum, and hippocampal formation (hippocampus, amygdala, entorhinal, and piriform cortices) were performed using (13)C and (1)H nuclear magnetic resonance spectroscopy and HPLC. In the hippocampal formation of epileptic rats, levels of glutamate, aspartate, N-acetyl aspartate, adenosine triphosphate plus adenosine diphosphate and glutathione were decreased. In all regions studied, labeling from [1,2-(13)C]acetate was similar in control and epileptic rats, indicating normal astrocytic metabolism. However, labeling of glutamate, GABA, aspartate, and alanine from [1-(13)C]glucose was decreased in all areas possibly reflecting neuronal loss. The labeling of glutamine from [1-(13)C]glucose was decreased in cerebral cortex and cerebellum and unchanged in hippocampal formation. In conclusion, no changes were detected in glial-neuronal interactions in the hippocampal formation while in cortex and cerebellum the flow of glutamate to astrocytes was decreased, indicating a disturbed glutamate-glutamine cycle. This is, to our knowledge, the first study showing that metabolic disturbances are confined to neurons inside the epileptic circuit.

High-resolution magic angle spinning MRS of breast cancer tissue.

High-resolution magic angle spinning (HR MAS) may develop into a new diagnostic tool for studying intact tissue samples, and several types of cancer have been investigated with promising results. In this study HR MAS spectra of breast cancer tissue from 10 patients have been compared to conventional high-resolution spectra of perchloric acid extracts of the same tissue type. The HR MAS spectra show resolution comparable to spectra of extracts, and two-dimensional techniques lead to identification of a majority of the constituents. More than 30 different metabolites have been detected and assigned. To our knowledge this is the most detailed assignment of biochemical components in intact human breast tissue. The spectra of intact breast cancer tissue differ from perchloric acid extracts by the presence of lipids and fewer signals in the low field region. HR MAS analysis of intact breast tissue specimens is a rapid method, providing spectra with resolution where relative quantification of the majority of the detected metabolites is possible.

Alterations in brain metabolism, CNS morphology and CSF dynamics in adult rats with kaolin-induced hydrocephalus.

The present study describes the biochemical changes, morphological development and the cerebrospinal fluid dynamics of the kaolin-induced hydrocephalus in the adult rat. Two, 4 and 6 weeks after microsurgical kaolin instillation into the rat cisterna magna the basal intracranial pressure and the cerebrospinal fluid outflow resistance were measured. To determine possible biochemical changes in the rat cerebrum, brain stem and cerebellum the concentrations of glutamine, glutamate, glutathione, aspartate, GABA, alanine and taurine were measured by high pressure liquid chromatography. In addition, ventriculomegaly and syringomyelia were assessed, measuring the lateral ventricles and central canals by means of an image-processing computer program. It could be shown that the acute phase of kaolin-induced hydrocephalus in the first 4 weeks is characterised by a high basal intracranial pressure, a considerably increased CSF outflow resistance and a rise in brain water content in the fourth week. The changes in the concentrations of amino acids were moderate. Glutamine was increased and taurine was decreased in the cerebrum and alanine was increased in the brain stem. The chronic phase, however, is defined by normal basal pressure, declining outflow resistance, progression of ventriculomegaly and distinct changes in the biochemical parameters such as a remarkable decrease of glutamate, glutamine and taurine in the cerebellum, a decrease of taurine and alanine plus an increase in glutamine in the cerebrum and an increase of alanine in the brain stem. Moreover, cerebral metabolism in the adult rat seems to be more resistant to the effects of hydrocephalus than metabolism in neonatal and infantile rats.