Neurotoxin effects on oxytocin, vasopressin and somatostatin in discrete rat brain areas

Monosodium-L-Glutamate (MSG) produces lesions to monoaminergic and peptidergic neurons in several brain areas. The present study examined the effect of neonatal MSG treatment on oxytocin (OXY), arginine-vasopressin (AVP) and somatostatin (SRIF) concentrations in several discrete brain areas of adult rats. OXY increased in the suprachiasmatic and arcuate nuclei and median eminence (ME) and decreased in the paraventricular nucleus of MSG-treated rats. MSG treatment caused AVP to increase in the arcuate nucleus and ME and decrease in the supraoptic nucleus. SRIF decreased following neonatal MSG treatment in both the ME and neurointermediate pituitary lobe. The results demonstrate that the effects of neonatal MSG treatment on neuropeptide content are not just limited to the arcuate nucleus. Furthermore, taken together with previous results, the data suggest that these changes may be indicative of functional deficits in the neuronal activity of some of these peptidergic neurons which, in turn, may be responsible for the abnormal secretion of several pituitary hormones observed in MSG-treated animals.     

Effects of neonatal administration of monosodium glutamate and castration on neurokinin A levels in the hypothalamus and anterior pituitary of rats.

The effects of neonatal administration of monosodium glutamate (MSG) and castration on hypothalamic and anterior pituitary levels of neurokinin A (NKA) were studied in male and female rats killed at 46 days of age. In male rats treated neonatally with MSG, body, anterior pituitary, testis, ventral prostate, and seminal vesicle weights and serum testosterone levels were significantly lower than in saline-injected controls. Hypothalamic NKA was significantly lower in MSG-treated male rats as compared with the controls, and no apparent changes were recorded in anterior pituitary NKA. Orchidectomy was followed by a significant decrease in hypothalamic NKA in saline controls, but not in MSG-treated rats. In female rats treated with MSG, there was a significant decrease in body, anterior pituitary, and ovarian weights, as compared with saline-injected controls, but no significant differences were observed in uterine weights and serum estradiol levels. Hypothalamic NKA was lower, although not significantly, in MSG-treated rats as compared with the respective controls, and no differences were recorded in anterior pituitary NKA levels. Ovariectomy was followed by a significant decrease in hypothalamic NKA in both MSG-treated and control rats, but NKA in the anterior pituitary was significantly increased after ovariectomy only in saline-treated controls, whereas MSG-treated females failed to show this response. It is concluded that neonatal MSG treatment resulted in a decrease of hypothalamic NKA, which was particularly pronounced in male rats without any significant change in anterior pituitary NKA levels. The response of hypothalamic NKA to castration and the response of anterior pituitary NKA to ovariectomy were also altered in MSG-treated rats; this may reflect a functional block of some neuroendocrine functions of the hypothalamus that resulted from the neuronal lesions induced by MSG.     

A decrease in thymus-mediated immune responses as a result of treatment of neonatal rats with glutamate

We investigated whether administration of monosodium l-glutamate (MSG) to neonatal rats would disrupt immune responses in intact and orchidectomized adult male rats. Neonatal male rats were treated with saline or MSG which causes severe endocrine abnormalities. Half of each group of animals were orchidectomized as adults and killed one week later along with intact rats. MSG treatment resulted in suppressed serum LH levels in intact rats. Thymus weight and spleen cellularity in intact animals were not affected by MSG treatment, but thymus weight increased within one week after orchidectomy in both saline- and MSG-treated groups. In intact rats, lymphocyte stimulation by the T cell specific mitogens (concanavalin A or phytohemagglutinin) or the B cell specific mitogen (lipopolysaccharide) was unaffected by prior treatment with MSG. However, MSG treatment blocked the decrease attributable to orchidectomy in concanavalin A and phytohemagglutinin stimulation of lymphocyte blastogenesis. The results suggest that administration of MSG to neonatal male rats can alter some immune responses in the adult animal.     

Potentiation of vasopressin analgesia in rats treated neonatally with monosodium glutamate

The analgesic response elicited by central administration of arginine vasopressin (AVP) appears to be dependent upon the integrity of the hypothalamic paraventricular nucleus (PVN), since lesions placed in the PVN eliminate AVP analgesia. A projection to the zona externa of the median eminence constitutes one of the VP-containing efferents of the PVN. Neonatal treatment with monosodium glutamate (MSG) destroys perikarya of the arcuate nucleus and median eminence. The present study examined whether AVP analgesia was affected in the MSG-treated rat and whether these alterations were accompanied by specific changes in VP immunoreactivity in the zona externa of the median eminence. Female rats, neonatally treated with either MSG or a saline control, were tested as adults on the tail-flick test following intracerebroventricular injections of 0, 75, 150 and 500 ng doses of AVP. After testing, selected animals were prepared for AVP and oxytocin immunocytochemistry of the median eminence. Significant potentiations in the magnitude of AVP analgesia were observed in MSG-treated rats. AVP and oxytocin immunoreactivity in the zona interna and oxytocin immunoreactivity in the zona externa of the median eminence were similar in MSG-treated and control rats. In contrast, AVP immunoreactivity in the zona externa of the median eminence was markedly reduced in the MSG-treated rat. These data suggest that VP analgesia may normally be inhibited by those medial-basal hypothalamic neurons affected by neonatal MSG treatment.     

Monosodium L-glutamate-induced convulsions: temporary alteration in blood-brain barrier permeability to plasma proteins.

Monosodium L-glutamate (MSG), a commonly used food additive, induces convulsive disorders in rats. A reversible change in the cerebrovascular permeability of plasma proteins occurs during convulsions induced by the intraperitoneal administration of 4.0 g/kg of MSG to the neonatal rat. During MSG-induced seizures, but not before or after, trypan blue dye enters into the brain tissues, whereas no dye penetration occurs in control rats receiving saline. The frequency of the incidence of MSG-induced convulsions is inversely proportional to the age of the animal. It decreases with the age of the rat. By 42 days of age no substantial seizure activity of dye penetration into the brain tissue occurs in MSG-treated rats. Histological examination indicates that seizure activity is not correlated with characteristic periventricular-arcuate area lesions known to be induced in neonates by parenteral MSG administration. No hypothalamic damage was observed in MSG-treated rats older than 10 days of age.     

Blockade of the selective increase in serum follicle-stimulating hormone concentration in immature female rats and its effects on ovarian follicular development

We investigated whether administration of monosodium L-glutamate (MSG) to neonatal female rats would block the selective increase in serum follicle-stimulating hormone (FSH) concentration in immature rats in an attempt to provide a model in which to study the importance of the selective FSH rise on ovarian follicular development. In two separate experiments, s.c. injections of MSG (4 mg/g BW) on Days 1, 3, 5, 7 and 9 after birth blocked the selective increase in serum FSH concentration observed on Days 7 and 15 without blocking basal FSH secretion. Serum luteinizing hormone (LH) levels were unaffected in the first experiment and changed little in the second. MSG-treated rats had smaller ovaries on Days 15 and 23. The ovaries of MSG-treated rats on Day 15 showed decreased follicular growth as evidenced by a decrease in the number and percentage of follicles with diameters greater than 50 microns, in the number of follicles with greater than 1 layer of granulosa cells, and in the number of follicles beyond the primary stage of follicular development. These differences between MSG-treated rats and controls all but disappeared by Day 23. The results demonstrate that neonatal administration of MSG blocks the selective increase in serum FSH concentration in immature female rats and suggest that this selective increase in serum FSH levels plays a role in the normal acceleration of ovarian follicular development but is not needed for the development of preovulatory follicles by the sixth week after birth.     

Effect of nitric oxide inhibition on blood pressure and corticosterone responses in adult rats neonatally treated with glutamate

The role of nitric oxide (NO) in the regulation of blood pressure and hypothalamic-pituitary-adrenal function of adult rats treated with monosodium glutamate (MSG) during the neonatal period was investigated. Blood pressure and the heart rate were registered by a computerized system of direct blood pressure measurement through an indwelling cannula in the femoral artery. The inhibition of the activity of NO synthase by acute injection of Nomega-nitro-L-argininemethylester (L-NAME, 30 mg/kg, i.v.) to control rats produced a rise of blood pressure and a fall of heart rate. Both responses were reduced in MSG-treated rats. Repeated administration of L-NAME (50 mg/kg, i.p, two times daily for 4 days) increased BP in both groups of animals. Corticosterone concentrations in the plasma were significantly increased in response to repeated L-NAME administration in MSG-treated rats, while ACTH levels were similar in both groups of animals. These data suggest that some of the cardiovascular and endocrine changes in rats treated with MSG may be due to the abnormal function of the NO system.     

Insulin-like growth factor I receptors in neuronal and glial cells. Characterization and biological effects in primary culture

Primary cultures of neuronal and glial cells from 1-day-old neonatal rats contain high affinity receptors for insulin-like growth factor I (IGF-I). The IC50 for displacement of 125I-IGF-I binding by unlabeled IGF-I was 3 nM for neuronal cells and 4 nM for glial cells. Unlabeled insulin was 20-50 times less potent. Apparent molecular mass of the alpha subunits of the IGF-I receptor was 125 kDa in neuronal and 135 kDa in glial cells. IGF-I induced autophosphorylation of the IGF-I receptor beta subunit in lectin-purified membrane preparations in a dose-dependent manner. The major phosphoamino acid of the beta subunit in both cell types was tyrosine in the IGF-I-stimulated state and serine in the basal state. Apparent molecular mass of the beta subunits of the IGF-I receptors was 91 kDa for neuronal and 95 kDa for glial cells. Tyrosine kinase activity of the IGF-I receptors was demonstrated by IGF-I-induced phosphorylation of the exogenous substrate poly(Glu, Tyr) 4:1 in both cell types. IGF-I had no effect on 2-deoxyglucose uptake in neuronal cells. In contrast, in glial cells, IGF-I stimulated 2-deoxyglucose uptake at very high doses, presumably acting via the insulin receptor. The effect of IGF-I as a neurotrophic growth factor in both neuronal and glial cells was demonstrated by its stimulation of [3H]thymidine incorporation. These findings suggest the IGF-I is an important growth factor in nervous tissue-derived cells.     

Effect of neonatal MSG treatment on day-night alkaline phosphatase activity in the rat duodenum

The day-night variation of food intake and alkaline phosphatase (AP) activity was studied in the duodenum of rats neonatally treated with monosodium glutamate (MSG) and saline-treated (control) rats. The animals were kept under light-dark conditions (light phase from 09:00 h to 21:00 h) with free access to food. AP activity was cytophotometrically analyzed in the brush-border of enterocytes separated from the tip, middle and cryptal part of the villi every 6 h over a 24-hour period. In comparison with the controls, MSG-treated rats consumed about 40% less food during the dark period and their 24-hour food intake was thus significantly lowered (P<0.001). On the other hand, the nocturnal feeding habit showed a similar pattern: food consumption was high during the night (65% vs. 75%) and the lowest consumption was found during the light phase (35% vs. 25%) in MSG-treated and control rats, respectively. In agreement with the rhythm of food intake, the highest AP activity was observed during the dark phase and was lowest during the light phase in both groups of animals. These significant day-night variations showed nearly the same pattern in the enterocytes of all observed parts along the villus axis. In comparison with the controls, a permanent increase of AP activity was observed in neonatal MSG-treated rats. This increase was more expressive during the dark phase of the day in the cryptal (P<0.001) and middle part of the villus (P<0.01). From the viewpoint of feeding, this enzyme in MSG-treated rats was enhanced in an inverse relation to the amount of food eaten i.e. despite sustained hypophagia the mean AP activity in the enterocytes along the villus axis was higher than in the control animals during all investigated periods. The present results suggest that the increased AP activity in MSG-treated rats is probably not a consequence of actual day-night eating perturbations but could be a component of a more general effect of MSG. This information contributes to better understanding of the function of intestinal AP and its relation to day-night feeding changes especially in connection with the MSG syndrome.     

MSG lesions decrease body mass of suckling-age rats by attenuating circadian decreases of energy expenditure

Suckling-age rats display endogenous circadian rhythmicity of metabolic rate (MR) with energy-saving, torpor-like decreases, which are sympathetically controlled and suppressed by leptin treatment. We investigated whether neonatal monosodium glutamate (MSG) treatment, known to cause arcuate nucleus damage and adult-age obesity, alters energy balance in the first two postnatal weeks. Continuously recorded MR and core temperatures (T(c)) show that MSG treatment disinhibits the periodic, sympathetically controlled, energy-saving drops of T(c) and MR. Increased energy expenditure thus explains reduced body fat at normal lean body mass found in MSG-treated pups artificially nourished identically to controls. In MSG-treated mother-reared pups, lean body mass is additionally reduced, suggesting that MSG also reduces suckling. Plasma leptin levels are similar in controls and MSG-treated pups but higher per unit of fat mass in the latter. We conclude that the postweaning development of MSG obesity and depressed thermogenesis are preceded by an early phase of increased energy expenditure with decreased fat deposition during suckling age and hypothesize cell damage in the arcuate nucleus to be involved in both.     

Effect of cholecystokinin on feeding is attenuated in monosodium glutamate obese mice

Treatment of newborn mice with monosodium glutamate (MSG) is neurotoxic for hypothalamic arcuate nucleus (ARC) and causes obesity. In the MSG-treated 16-week-old NMRI mice, we detected specific ablation of ARC neuronal cells, 8 times higher fat to body mass ratio but unchanged body mass compared to controls, advanced hyperglycemia and hyperinsulinemia--both more pronounced in males, and hyperleptinemia--more severe in females. After fasting, the MSG-treated mice showed attenuated food intake compared to controls. Cholecystokinin octapeptide, which decreased food intake in a dose-dependent manner in 24 h fasted controls, did not significantly affect food intake in the MSG-treated animals. We propose that the obesity-related changes in the feeding behavior of the MSG-treated obese mice were the result of missing leptin and insulin receptors in ARC and consequent altered neuropeptide signaling. This makes the MSG model suitable for clarifying generally the central control of food intake.     

Effect of fasting and refeeding on duodenal alkaline phosphatase activity in monosodium glutamate obese rats

In the present work the effects of fasting and refeeding on fat pad weight and alkaline phosphatase activity in the brush border of individual duodenal enterocytes have been evaluated in male Wistar rats with obesity induced by monosodium glutamate (MSG) treatment during the early postnatal period. Neonatal rats were treated subcutaneously with MSG (2 mg/g b.w.) or saline (controls) for 4 days after birth. At 4 months of age, two types of experiments were performed. In the first experiment rats, were submitted to 3 or 6 days lasting food deprivation. In the second experiment the rats were refed for 3 or 6 days ad libitum or restrictedly (60% of pre-fasting intake) after a 6 day-fasting period. Fasting and refeeding influenced the body fat and function of the duodenum in MSG-treated rats differently as compared to the controls. However, alkaline phosphatase activity and the weight of epididymal and retroperitoneal fat depots were significantly increased in MSG obese rats (P<0.001) during all the periods examined. While 3 days of food deprivation resulted in both groups in a similar loss of adipose tissue weight and alkaline phosphatase activity, the decrements of these parameters after 6 days of fasting were lower in obese rats suggesting that their capacity to spare body fat stores was enhanced. After 3 days of ad libitum refeeding, a more marked adaptational increase of food consumption and also a significantly increased alkaline phosphatase activity above the pre-fasting level (P<0.01) was observed in the MSG-treated rats. Consequently, a more rapid body fat restoration was demonstrated in these animals. Refeeding of rats at 60% of the pre-fasting intake level resulted in a significant increase of alkaline phosphatase activity in both the MSG and control group; moreover, as food restriction continued, MSG-treated rats tended to further increase the enzyme activity. Our results revealed that MSG treatment of neonatal rats may significantly change the intestinal functions. Permanently increased alkaline phosphatase activity observed in MSG obese rats during all investigated periods suggests that this functional alteration is probably not a consequence of actual nutritional variation but could be a component of regulatory mechanisms maintaining their obesity at critical values.     

Effect of monosodium glutamate treatment during neonatal development on lipogenesis rate and lipoprotein lipase activity in adult rats.

Monosodium glutamate (MSG) has been shown to alter several neuroendocrine functions in neonatally treated rats. To evaluate possible alterations in lipogenesis rate and lipoprotein lipase (LPL) activity, male and female rats were injected during the neonatal period with MSG or saline (controls). In male MSG rats, an increase in lipogenesis of liver and retroperitoneal adipose tissues was observed. Triton WR 1339 (an LPL inhibitor) administration decreased retroperitoneal lipogenesis in these animals. In female rats, MSG-treatment increased lipogenesis only in gonadal and retroperitoneal adipose tissues. No change was observed in hepatic lipogenesis and the Triton administration did not change retroperitoneal lipogenesis. LPL activity was increased in the gonadal and retroperitoneal adipose tissues in male and female MSG-treated rats. These data suggest that there is a specific sex-dependent response in the development of MSG-induced obesity.     

Late effects of postnatal administration of monosodium glutamate on insulin action in adult rats

Early postnatal administration of monosodium glutamate (MSG) to rats induces obesity, hyperinsulinemia and hyperglycemia in adulthood, thus suggesting the presence of insulin resistance. We therefore investigated the effects of insulin on glucose transport and lipogenesis in adipocytes as well as insulin binding to specific receptors in the liver, skeletal muscle and fat tissues. An increase of plasma insulin, glucose and leptin levels was found in 3-month-old rats treated with MSG during the postnatal period. The attenuation of insulin stimulatory effect on glucose transport was observed in MSG-treated rats. Despite the lower basal and insulin-stimulated glucose uptake, the incorporation of glucose into lipids was significantly higher in MSG-treated rats, suggesting a shift in glucose metabolism towards lipid synthesis in fat tissue. Insulin binding to plasma membranes from the liver, skeletal muscle and adipocytes was decreased in MSG-treated rats. This is in agreement with the lower insulin effect on glucose transport in these animals. Furthermore, a decreased amount of GLUT4 protein was found in adipocytes from MSG-treated obese rats. The results demonstrated an attenuation of insulin effect on glucose transport due to a lower insulin binding and lower content of GLUT4 protein in MSG-treated rats. However, the effect of insulin on lipogenesis was not changed. Our results indicated that early postnatal administration of MSG exerts an important effect on glucose metabolism and insulin action in adipocytes of adult animals.     

Inhibition of VEGFR-2 by SU5416 increases neonatally glutamate-induced neuronal damage in the cerebral motor cortex and hippocampus

Vascular endothelial growth factor (VEGF) exerts neuroprotective or proinflammatory effects, depending on what VEGF forms (A–E), receptor types (VEGFR1–3), and intracellular signaling pathways are involved. Neonatal monosodium glutamate (MSG) treatment triggers neuronal death by excitotoxicity, which is commonly involved in different neurological disorders, including neurodegenerative diseases. This study was designed to evaluate the effects of VEGFR-2 inhibition on neuronal damage triggered by excitotoxicity in the cerebral motor cortex (CMC) and hippocampus (Hp) after neonatal MSG treatment. MSG was administered at a dose of 4 g/kg of body weight (b.w.) subcutaneously on postnatal days (PD) 1, 3, 5, and 7, whereas the VEGFR-2 inhibitor SU5416 was administered at a dose of 10 mg/kg b.w. subcutaneously on PD 5 and 7, 30 min before the MSG treatment. Neuronal damage was assessed using hematoxylin and eosin staining, fluoro-Jade staining, and TUNEL assay. Additionally, western blot assays for some proteins of the VEGF-A/VEGFR-2 signaling pathway (VEGF-A, VEGFR-2, PI3K, Akt, and iNOS) were carried out. All assays were performed on PD 6, 8, 10, and 14. Inhibition of VEGFR-2 signaling by SU5416 increases the neuronal damage induced by neonatal MSG treatment in both the CMC and Hp. Moreover, neonatal MSG treatment increased the expression levels of the studied VEGF-A/VEGFR-2 signaling pathway proteins, particularly in the CMC. We conclude that VEGF-A/VEGFR-2 signaling pathway activation could be part of the neuroprotective mechanisms that attempt to compensate for neuronal damage induced by neonatal MSG treatment and possibly also in other conditions involving excitotoxicity.     

Decreased ketonaemia in the monosodium glutamate-induced obese rats

Plasma concentrations of total ketone bodies, acetoacetate (AcAc) and 3-hydroxybutyrate (3-OHBA) in monosodium glutamate (MSG)-induced obese rats were measured. MSG-treated rats showed higher Lee's indices, shorter naso-anal and tail length, and a more marked intraperitoneal fat deposition than control rats. Plasma concentrations of glucose, free fatty acid, triglyceride and phospholipids were significantly increased in the MSG-treated rats as compared to the control rats (24 weeks-old). Plasma levels of total ketone bodies, AcAc and 3-OHBA were all decreased in the MSG-treated rats as compared to control rats. The ratio, 3-OHBA/AcAc in the MSG-treated rats were not different from those in the control rats.     

Steroid-monoamine feedback interactions in discrete brain regions using as a model the monosodium glutamate (MSG)-lesioned rat

The present studies examine the effects of neonatal treatment with monosodium glutamate (MSG) on dopamine (DA), 5-hydroxytryptamine (5-HT) and norepinephrine (NE) metabolism in discrete brain regions and correlate them with steroid receptor kinetics in the anterior pituitary (PIT), preoptic hypothalamus (POA) and caudal hypothalamus (HYP), and with steroid negative and positive feedback effects on luteinizing hormone (LH) secretion. Substantial decreases in the neuronal activity of all three amines in the arcuate nucleus, decreased DA and 5-HT metabolism in the suprachiasmatic nucleus and, surprisingly, increased metabolism of 5-HT and NE in the median eminence was observed in adult ovariectomized (OVX), MSG-treated versus OVX, vehicle-treated litter mate controls. Measurement of estradiol receptors in the nuclear and cytosolic fractions of the POA, HYP and PIT from MSG- and vehicle-treated rats killed during diestrus or 2 weeks after OVX revealed no differences. Similarly, no differences in cytosolic progestin receptors between control and MSG unprimed or estradiol-primed, OVX rats or on progestin receptor translocation induced by progesterone in Eb-primed rats were observed. Negative and positive feedback effects of estradiol or the positive feedback of progesterone on LH secretion were not significantly impaired in MSG rats, and indeed, MSG animals actually were hyper-responsive to the administration of the steroids or of luteinizing hormone-releasing hormone. These results indicate that the MSG-induced damage to DA, 5-HT and NE elements observed within several preoptic and hypothalamic nuclei does not impair estrogen and progestin receptor kinetics, nor does it prevent adequate negative or positive steroid feedback responses, if appropriate steroid regimens are employed, and that the impaired gonadal function reported in these animals does not result primarily from inadequate steroid feedback mechanisms.     

Role of astrocytes and altered regulation of spinal glutamatergic neurotransmission in stress-induced visceral hyperalgesia in rats

Glutamate (Glu) is the primary excitatory neurotransmitter in the central nervous system and plays a critical role in the neuroplasticity of nociceptive networks. We aimed to examine the role of spinal astroglia in the modulation of glutamatergic neurotransmission in a model of chronic psychological stress-induced visceral hyperalgesia in male Wistar rats. We assessed the effect of chronic stress on different glial Glu control mechanisms in the spinal cord including N-methyl-d-aspartate receptors (NMDARs), glial Glu transporters (GLT1 and GLAST), the Glu conversion enzyme glutamine synthetase (GS), and glial fibrillary acidic protein (GFAP). We also tested the effect of pharmacological inhibition of NMDAR activation, of extracellular Glu reuptake, and of astrocyte function on visceral nociceptive response in naive and stressed rats. We observed stress-induced decreased expression of spinal GLT1, GFAP, and GS, whereas GLAST expression was upregulated. Although visceral hyperalgesia was blocked by pharmacological inhibition of spinal NMDARs, we observed no stress effects on NMDAR subunit expression or phosphorylation. The glial modulating agent propentofylline blocked stress-induced visceral hyperalgesia, and blockade of GLT1 function in control rats resulted in enhanced visceral nociceptive response. These findings provide evidence for stress-induced modulation of glia-controlled spinal Glu-ergic neurotransmission and its involvement in chronic stress-induced visceral hyperalgesia. The findings reported in this study demonstrate a unique pattern of stress-induced changes in spinal Glu signaling and metabolism associated with enhanced responses to visceral distension.