Neonatal administration of monosodium glutamate (MSG) produces pathological lesions in many brain regions. There are indications that MSG treatment could also influence the neurons of the hypothalamic paraventricular nucleus (PVN). The goal of this study was to find out whether MSG treatment could alter the activity of the corticotropin-releasing hormone synthesizing neurons, i.e. the principal regulators of the corticotropin hormone secretion, located in the medial posterior subdivision of the PVN. The activity of CRH neurons was assessed by changes in CRH mRNA levels in response to both stimulatory and inhibitory conditions induced by immobilization and water deprivation, respectively. In addition, effect of the circulating glucocorticoid deficit induced by bilateral adrenalectomy was investigated. The obtained data show that in MSG-treated animals the rise in CRH mRNA in response to immobilization stress and adrenalectomy as well as the decrease after water deprivation were similar to the changes seen in controls. In addition POMC mRNA changes in MSG-treated animals indicate an uninterrupted capability of CRH neurons to transform different signals to corticotropin cells. it can be concluded that CRH neurons of the PVN are not functionally altered, in spite of the widespread neurotoxic effect of MSG treatment. [Neurol Res 1999; 21: 775-7801
Keywords: Monosodium glutamate; hypothalamic paraventricular nucleus; adrenalectomy; water deprivation; immobilization stress, CRH mRNA, POMC mRNA
Neonatal administration of monosodium glutamate (MSG) produces pathological lesions in many brain regions 4 . In the hypothalamus, the arcuate nucleus has been reported to be the most affected brain area displaying partial or complete loss of cell bodies containing immunoreactivity for growth hormonereleasing factor, galanin, dynorphin, enkephalin, corticotropin-like intermediate peptide, neuropeptide Y, and neuropeptide K5,6. Marked decreases in the content of different neuropeptides have also been demonstrated in the median eminence.5
There are suggestions that MSG treatment could also influence the activity of neurons located in the hypothalamic paraventricular nucleus (PVN Pronounced alterations have been described in the metabolism and content of hypothalamic neurotransmitters8,9 , as well as in occurrence of specific degenerations in the hippocampus10, i.e. a higher regulatory center of the PVN. Corticotropin-releasing hormone (CRH) synthesizing neurons occupy the medial posterior subdivision of the PVN and provide the stimulus for the release of adrenocorticotrophic hormone (ACTH)11. Activity of CRH neurons is under the control of both stimulatory and inhibitory neural inputs and a negative feedback control established by glucocorticoids12. Findings of hyper-responsivity of some PVN neurohormones to stimulation by KCI 13 indicate that neonatal MSG-treatment can destroy inputs to certain population of PVN neurons and thus induce an alteration in their function.
Recently, we have provided evidence that MSG treatment does not impair the peripheral responses of the HPA axis to acute stress reflected by normal magnitude of ACTH and corticosterone responsiveness". A natural assumption would be that these responses are mediated by stress activated CRH neurons. However, the control of ACTH secretion is multifactorial and thus the MSG neurotoxicity could alter the sensitivity of CRH neurons to different stressors. Therefore the aim of the present study was to elucidate the level of susceptibility of CRH neurons. The activity of CRH neurons was assessed by the changes in CRH mRNA levels in response to both stimulatory and inhibitory conditions induced by immobilization and water deprivation, respectively. In addition, effect of circulating glucocorticoid deficit induced by bilateral adrenalectomy was investigated in MSG-treated animals. As additional physiological parameters, pituitary POMC mRNA, plasma ACTH and corticosterone levels were analyzed.
MATERIALS AND METHODS
Animals and in vivo -Procedures Male offsprings of Sprague-Dawley rats were used (Charles River Wiga, Silzfeld, Germany) housed three or four per cage under standard conditions of lighting (light on from 0600 h-1800 h) and temperature 230 +/-2degC, with free access to food and water.
MSG-treated animals were injected intraperitoneally O.p.) with MSG (4 mg g^sup -1^ body weight, Merck, Darmstadt, Germany) dissolved in 0.9% NaCl or equivalent volumes of 10% NaCl iso-osmotic to MSG solution (littermate controls) on post-birth days 2, 4, 6, 8 and 10. The animals were weaned at the age of 21 days and used at age of 10-14 weeks.
Immobilizaiton of the animals was performed in a prone position by inserting their heads through steel wire loops fixed on a board fastening their limbs to metal strips with adhesive tape1 5.
In the water-deprived animals, water was removed at 0800 h and 72 h later the animals were sacrificed by decapitation for blood sampling and tissue collection.
Bilateral adrenalectomy or sham operation was performed through a dorsal approach under pentobarbital anesthesia two or seven days before tissue collection. Adrenalectomized rats had free access to 0.9% saline.
All experiments were performed in conscious freely moving rats. The selected brains and pituitaries for mRNA measurements by in situ hybridization were immediately frozen in isopentane at -30degC or dry ice and stored at -70degC. The blood samples were collected into cooled polytethylene tubes with EDTA as anticoagulant and plasma was stored at -20degC until analyzed.
Hormone measurements and statistical analysis
Plasma ACTH was analyzed by a radioimmunoassay as described previously16. The specific antibody was kindly provided by G.B. Makara (Budapest, Hungary). Corticosterone was measured in dichloromethane extracts of plasma (10 (mu)l) by a radioimmunoassay according to the previously described procedure17.
Data were statistically evaluated by one way analysis of variance (ANOVA) followed by Scheffe or Fisher tests. When comparisons were performed for two groups only, unpaired Student's t-test was used.
In situ hybridization
Hybridization was performed on 12 (mu)m thick sections cut on a cryostat and thaw-mounted onto poly-L-lysinecoated slides using [^sup 35^S]deoxy-ATP labeled (1200Ci mmol^sup -1^; NEN, DuPont, Boston, MA, USA) 48-mer oligonucleotides by terminal deoxynucleotidyl transferase (Boehringer Mannheim GmbH, Wien, Austria) with a specific activity 12x10^sup 6^CpMpmol^sup -1^. The probes were complementary to the bases corresponding to amino acids 22-37 of rat/human pro-CRH, and 102117 of rat POMC (a gift from Dr G. Aguilera, USA), synthesized by Synthecell (Rockville, MID, USA). The procedure was performed essentially as previously described .14
In each experiment all control and experimental groups were processed in the same hybridization procedure and exposed to Hyperfilm-beta max (Amersham, Piscataway, NJ, USA). Optical densities of the autoradiographic hybridization signals were quantitated using a computerized image analysis system (imaging Research, Inc., St. Catherines, Ontario, Canada). The comparison between the individual groups was performed after subtracting the background signal from the values at least 6 sections animal-' in a minimum of 4 rats group^sup -1^.
General effects of MSG
As expected, neonatal treatment with MSG resulted in several characteristic features such as decreases in body (by 19.5%), adrenal (by 18%) and pituitary (by 52%) weight (Table 1). Degenerated optic nerves, reduction of the optic chiasm size and stunted growth and obesity were also noticeable in these animals.
Effect of acute immobilization
Basal morning levels of CRH mRNA in the PVN of control and MSG-treated animals exhibited autoradiographic signals of similar optical density. Animals exposed to 120 min of acute immobilization OMO) and sacrificed 3 h later also exhibited a similar pattern of elevations of CRH mRNA levels in the PVN (Figure 1).
On the other hand, optical density of basal levels of POMC mRNA was significantly higher in MSG-treated animals in comparison with controls. This difference was also noticeable in their response to acute IMO stress (Figure 2).
Effect of water deprivation
The optical density of the hybridization signal for basal CRH mRNA levels in the PVN of MSG-treated animals did not differ significantly from the signal measured in the control group of animals. Water deprivation for 72 h significantly lowered CRH mRNA levels not only in the controls but also in the MSGtreated rats. This decrease was almost of the same magnitude in both investigated groups (Figure 3).
Morning measurements of basal plasma levels of corticosterone were similar in control and MSG-treated rats (4.68+/-2.3(mu)gdl^sup -1^ vs. 3.38+/-0.9(mu)gdl^sup -1^). Water deprivation markedly elevated plasma corticosterone levels in both control and MSG animals (12.88+/-2.5(mu)gdl^sup -1^ vs. 8.12+/-2.0(mu)gdl^sup -1^), however, the increase in MSG-treated rats did not reach significance. Plasma ACTH levels were not influenced by MSG treatment or water deprivation for 72 h (results not shown). The plasma osmolality was significantly elevated in both groups of animals (C 316+/-1.8 vs. MSG 316+/-0.2rrvOsmol l^sup -1^ and C 345+/-3.4 vs. MSG 339+/- 6.8 mOsmol l^sup -1^).
Effect of adrenalectomy
Removal of endogenous glucocorticoids by bilateral adrenalectomy for two days resulted in a clear increase of CRH mRNA levels in the PVN not only in the control but also in the MSG pretreated group of rats (Figure 4). On the seventh day of adrenalectomy, no additional increase in the levels of CRH mRNA was observed in controls or MSG-treated rats (Figure 4).
In contrast to CRH mRNA, a progressive increase was seen in anterior pituitary POMC mRNA levels. Two days after adrenalectomy, POMC mRNA levels were doubled and were elevated about four times after seven days. On the seventh day, the values were significantly higher compared to those on day 2 of adrenalectomy (Figure 5).
In sham operated MSG-treated rats, significantly higher POMC mRNA levels were found compared to those in sham operated controls. Similarly as in controls, adrenalectomy induced a progressive rise in POMC mRNA levels. Although responses of MSG-treated animals to adrenalectomy were significantly higher in comparison with those of controls, the increments did not differ significantly from each other.
Basal circulating levels of corticosterone were significantly elevated in sham-operated MSG-treated animals (5.48+/-1.6(mu)qdl^sup -1^) in comparison with controls (0.43+/-0.15(mu)gdl^sup -1^), while basal peripheral levels of ACTH showed no difference. Likewise in the case of POMC mRNA, adrenalectomy produced a trend of progressive elevation in ACTH levels in both groups of animals (Figure 6).
The results of the present study demonstrate that the functional integrity of CRH neurons of the medial posterior subpopulation of the PVN in response to several different experimental conditions was not altered by neurotoxic lesions induced by neonatal treatment with MSG.
In adult rats variety of stressors, such as hypertonic saline injection18, insulin-induced hypoglycemia19, restraint and swimming stress20-22 are capable of inducing an increase in CRH mRNA levels in the PVN. Immobilization stress has also been shown to produce a marked up-regulation of PVN CRH mRNA levels 22 which correlates with increased pituitary POMC mRNA expression and elevations of plasma ACTH 23,24 and corticosterone levels. In the present experiments, MSG treated animals showed the same pattern of responsiveness to immobilization stress as the controls, which may account for an unaltered capability of CRH neurons of MSG animals to perceive and to process by stress evoked signals. In contrast to the similarity seen in basal CRH mRNA levels, the basal POMC mRNA expression was significantly higher in MSG-treated animals, which is in accordance with our previous findingS14. Despite these differences, the responsiveness of POMC mRNA to immobilization was almost identical in both groups of animals reflected by the same magnitude of the increment values. Finally, this parallelism in the POMC mRNA response to immobilization corresponds well with plasma ACTH and corticosterone levels indicating an uninterrupted capability of CRH neurons to transform stimulatory inputs towards the hypophyseal corticotrops.
Osmotic conditions elicited by water deprivation or 2% saline intake, which lead to a marked activation of the magnocellular vasopressinergic neurons and an increase in plasma osmolality and vasopressin levels, are accompanied by a reduction of CRH mRNA levels in the hypophysiotropic neurons of the PVN 25,26. The factors responsible for CRH mRNA reduction are not known and the possibility of involvement of high circulating levels of glucocorticoids has not been proved yet. Some non-osmotic conditions such as lactation27, inflammatory stress28 and food deprivation29,30 may also induce an inhibition of CRH mRNA expression in the PVN. None of them interfered with the inhibitory effect of water deprivaiton, with the exception of food deprivation, which elicits inhibition of weaker magnitude29. Reduced expression of CRH mRNA in water deprived MSG-treated animals suggests an unaltered osmotic circuitry mediating inhibitory signals to the CRH neurons in the PVN. Notably, in the salt loaded animals, the interruption of the connection between the lamina terminalis and PVN area re-established the osmotically inhibited CRH mRNA signal in the hypophysiotropic population of CRH neurons31.
CRH neurons in the PVN display high sensitivity to the lack of circulating glucocorticoids by a general upregulation of the neuronal activity manifested, among others, by promotion of a gradual rise in CRH mRNA levelS32. Maximal rise in CRH mRNA levels was reported to occur four days after adrenalectomy reaching a plateau lasting up to at least seven days. The present data show that this plateau and apparently maximal CRH mRNA hybridization signal in the PVN is achieved already on the second day of adrenalectomy.
MSG treatment did not alter basal expression of CRH mRNA in any of the presented experiments which is in accordance with our previously published data14 . The prompt and progressive activation of adenohypophyseal POMC mRNA expression seen two and seven days after adrenalectomy in sham controls is consistent with the literature data 33 and it is due to the activation of hypophysotropic CRH neurons manifested by not only an increased CRH gene expression and subsequent elevation of CRH mRNA levels but also by an increased secretion of CRH neuropeptide into the portal circulation followed by stimulation of adenohypophyseal corticotrops and secretion of ACTH 24 . Thus, the elevations of the hypothalamic CRH mRNA, pituitary POMC mRNA and plasma ACTH levels in MSG-treated animals observed in the present study indicate not only the preservation of normal sensitivity of CRH neurons to the lack of circulatory glucocorticoids but also the existence of a functionally uninterrupted link between the hypophysiotropic CRH population of neurons and pituitary corticotrops.
From the present study it is evident that in MSG-treated rats, CRH neurons of the PVN are not influenced and functionally altered, in spite of the widespread neurotoxic effect of neonatal monosodium glutamate treatment. However, a dysfunction of other neuronal phenotypes present in the PVN cannot be excluded.
We thank Dr G. Aguilera, NIH, Bethesda, for providing oligonucleotide probes and Dr G.B. Makara, Budapest, Hungary, for the ACTH antibody. This work was supported by grants of EC (CIPACT 930227) and VEGA 2/6084.
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Alexander Kiss, Ivana Skultetyova and Daniela Jezova
Institute of Experimental Endocrinology, Slovak Academy of Sciences, Bratislava, Slovakia
Correspondence and reprint requests to: Dr Alexander Kiss, Institute of Experimental Endocrinology, Slovak Academy of Sciences, Vlarska 3, 833 06 Bratislava, Slovakia. Accepted for publication June 1999.
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