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Age- and dose-dependent effects of neonatal monosodium glutamate (MSG) administration to female rats
Neurotoxicologyand Teratology, Vol. 11, pp. 331-337. ©Pergamon Press plc, 1989. Printed in the U.S.A.
0892-0362/89 $3.00 + .00
Age- and Dose-Dependent Effects of Neonatal Monosodium Glutamate (MSG) Administration to Female Rats R. D A W S O N , J R . , J. W. S I M P K I N S A N D D. R. W A L L A C E
Department of Pharmacodynamics, University of Florida, College of Pharmacy JHMHC, Box J-487, Gainesville, FL 32610 R e c e i v e d 22 July 1988
DAWSON, R., JR., J. W. SIMPKINS AND D. R. WALLACE. Age- and dose-dependent effects of neonatal monosodium glutamate (MSG) administration to female rats. NEUROTOXICOL TERATOL 11(4) 331-337, 1989.--The age- and dose-dependent effects of neonatal MSG were evaluated in pre- and postpubertal female rats. The neurotoxic action of MSG was assessed by examining monoamine content in microdissected regions of the mediobasal hypothalamus. MSG was administered at a dose of 4 mg/g on postnatal days 2 and 4 (MSG-Lo) or on postnatal days 2, 4, 6 and 8 (MSG-Hi). MSG-Hi treatment significantly reduced dopamine (DA) content in the arcuate nucleus (ANH) and lateral median eminence (LME) on postnatal day 21 when compared to NaCl-injected controls. DA content relative to controls was not altered in the ANH or LME postnatal or postnatal day 60 in MSG-Hi, however, norepinephrine (NE) was significantly (p<0.05) decreased on both postnatal day 21 and 60 in the LME. MSG-Lo treatment significantly (p<0.05) reduced ANH NE content on postnatal day 60 compared to controls. Both MSG-Hi and MSG-Lo treatment increased 5-hydroxyindoleacetic acid content in the preoptic area (POA) on postnatal day 60 relative to the controls. Developmental changes independent of MSG treatment were noted in the hypothalamus. DA and 3,4-dihydroxyphenylacetic acid (DOPAC) content in the POA were 2-3-fold higher on postnatal day 21 compared to postnatal day 60. In contrast, DA content increased with age in the ANH, LME and medial ME. NE content in the ANH increased as a function of age in controls, but not in MSG-treated rats, The effects of MSG treatment on the postnatal development and maturation of neurons in the mediobasal hypothalamus were discussed in relation to the direct neurotoxicity of MSG. Monosodium glutamate Excitotoxin
Arcuate nucleus
Developmental neurochemistry
Dopamine
Median eminence
The DA neurons that project to the medial portion of the median eminence appear distinct in functional characteristics from the DA neurons that project to the lateral median eminence (35,49). Median eminence DA content has been reported to be decreased (38) or unaltered (24) by MSG treatment. Basal prolactin levels are usually unaltered by MSG treatment (7,38), although hyperprolactinemia has been reported (21,47). LH regulation is generally reported to be disrupted by MSG treatment (7, 18, 24, 52) and gonadal atrophy is a hallmark of the MSG syndrome (11, 40, 46). Thus, there appears to be a greater impact of MSG treatment on neuronal systems projecting to the lateral median eminence (LME), which include LH releasing hormone (LHRH) containing fibers (20,30), in comparison to the medial median eminence (MME) which appears important in mediating the tonic dopaminergic inhibition of prolactin release (37). The aim of the present study was to examine the effects of MSG treatment on the postnatal neurochemical development of discrete mediobasal hypothalamic regions including the LME and MME at pre- and postpubertal time points. A second aim was to examine the dose-response relationship of MSG treatment by employing two different dosing regimens, since the timing of injections, as well as the dose, may alter the functional consequences of MSG treatment. The pronounced ontogenic changes
THE developmental profile of mediobasal hypothalamic monoamine metabolism has previously been reported to be altered by neonatal MSG treatment (13). The arcuate nucleus of the hypothalamus (ANH) is a major target for the neurotoxic action of MSG and the neuronal loss that accompanies MSG treatment results in numerous neuroendocrine deficits involving metabolic, reproductive and growth disturbances (3, 9, 34, 36, 38, 46). Previous studies indicate that at birth the ANH is in an immature state of histogenesis and undergoes an extensive postnatal development (8, 26, 31, 32). The ANH undergoes progressive structural maturation during the sexual differentiation of the hypothalamus (1, 20, 33, 56). Significant organizational and synaptogenic events occur during the ontogenetic development of the ANH as a prelude to full development of a functional hypothalamic-pituitary axis. MSG treatment appears to alter the ontogenesis of ANH dopamine (DA) neurons (8,32) and evokes reorganization of neuroendocrine centers regulating pulsatile luteinizing hormone (LH) secretion (48). MSG may, therefore, exert its effects both by direct excitotoxic mechanisms and also indirectly by altering the postnatal ontogenic development of neuronal and synaptic elements in the mediobasal hypothalamus. Anatomical and neurochemical data suggest that the DA neurons of the ANH exhibit functional heterogeneity (8, 11, 49).
331
332
DAWSON, SIMPKINS AND WALLACE
occurring as a result of the postnatal and sexual maturation of the ANH make this region an especially vulnerable target for the neurotoxic actions of MSG.
ARCUATE NUCLEUS 50.
I---] CONTROL"
I
40.
METHOD
+J 30-
~
HIGH
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~.~.
Animals
,
20-
Timed pregnant Sprague-Dawley rats were obtained from Charles River Breeding Laboratory (Wilmington, MA) and their offspring served as subjects in this experiment. MSG (400 mg/ml in distilled water) was administered subcutaneously at a dose of 4 mg/g on postnatal days 2 and 4 (MSG-Lo) or postnatal days 2, 4, 6 and 8 (MSG-Hi). Littermates were injected subcutaneously with 2.0% sodium chloride as a hypertonic control. All rat pups were randomly assigned to dams after the injection procedure on postnatal day 2 and were cross-fostered until weaning on postnatal day 21. One group of female MSG-treated and control rats were killed by decapitation on postnatal day 21 and the brains were rapidly removed and placed on dry ice. The frozen brains were stored at - 8 0 ° C until processing for microdissection by a modification (17) of the method of Palkovits (44). The remaining female rats were group housed according to treatment condition and checked daily for the time of vaginal opening. Vaginal smears were taken daily by lavage after vaginal opening until postnatal day 60. On postnatal day 60, the rats were killed by decapitation and the brains were processed as described above. Body weights and organ weights were determined in both 21- and 60-day-old rats. All rats were maintained on a 12-hour light-dark cycle with food and water available ad lib.
Neurochemistry Tissue punches were collected in 1.5-ml microcentrifuge tubes containing 40 p~l of 0.1 M perchloric acid which contained 100 mg/1 Na2EDTA and 1.0 mM glutathione. Samples were homogenized by sonication (Heat Systems-Ultrasonics, Plainview, NY) and centrifuged for 4 minutes in a Beckman microfuge (Palo Alto, CA). The supernatant was diluted 1:1 with mobile phase and analyzed for monoamine and metabolite content using high performance liquid chromatography with electrochemical detection (25). The limits of detection for monoamine and metabolite content were approximately 20 pg/20 txl injection at a detector sensitivity of 1.0 nA. Protein content of the tissue pellet was determined using the method of Bradford (4). Data were subjected to two-way analysis of variance (ANOVA) to determine the overall effect of age and MSG treatment and any age x dose interactions. Individual planned comparisons were performed using the least significant difference (LSD) test (16).
t.u z 10-
2420. ~16+1
~
4-
21
60 DAY
FIG. 1. DA and NE content in the ANH of 21- and 60-day-old female MSG-treated and control rats. *p<0.05 MSG-treated versus control; Op<0.05 MSG-Hi versus MSG-Lo.
neonatal MSG treatment on postnatal day 60. There was an overall significant (p<0.001) age-related increase in MME DA content. The DA metabolite, 3,4-dihydroxyphenylacetic acid (DOPAC), was not detectable in 21-day-old rats, but was detectable in the MME at postnatal dz.y 60 (Table 1). No differences in MME DOPAC were found between MSG-treated and control rats. There were no age- or treatment-related alterations in MME norepinephrine (NE) (Fig. 3). There were significant (/7<0.01) age and treatment effects on DA content in the LME (Fig. 2). DA content in MSG-Hi rats was significantly (p<0.05) lower than controls or MSG-Lo on post-
MEDIAL ME
LATERAL ME
320. 280
RESULTS DA content in the ANH was significantly decreased in MSG-Hi rats on postnatal day 21 when compared to control or MSG-Lo rats (Fig. 1). There was a significant (p<0.001) effect of age on DA content in the ANH, although only the MSG-Hi rats exhibited a significant increase in ANH DA content from postnatal day 21 to 60. MSG treatment significantly (p<0.05) decreased ANH NE content (Fig. 1). No significant changes in ANH NE were present on postnatal day 21, but the significant (p<0.001) age-related increase in ANH NE was attenuated in MSG-treated rats. MSG-Lo rats had significantly (p<0.05) lower ANH NE content than controls on postnatal day 60 (Fig. 1). There was no effect of MSG treatment on MME DA content (Fig. 2). The high variability in the control group on day 21 may have obscured a real depletion in MME DA in the MSG-Hi group. MME DA was not altered by
12, .
]
CONTROL
]
LOW
~ 1 HIGH
240
ram200
~ o.
El
160.
.< 120a
El
804021
60
21
60
DAY
FIG. 2. DA content m the MME and LME of MSG-treated and control rats. *p<0.05 MSG-treated vs. control; Op<0.05 MSG-Hi vs. MSG-Lo.
DEVELOPMENTAL EFFECTS OF M S G
333
TABLE I
TABLE 2
DOPAC LEVELS IN THE HYPOTHALAMUS OF MSG-TREATED AND CONTROL RATS
MONOAMINE AND METABOLITE CONTENT IN THE PREOPTIC AREA
Region
Control
MSG-Lo
AN Day 21 Day 60
9.4 ± 11.9 ±
MME Day 60
43.4 ± 10.0
1.5 1.5
9.5 ± 8.8 ±
MSG-Hi
1.6 0.7
10.5 ± 0.5 10.0 ± 0.9
44.5 ± 10.5
49.7 ± 6.66
LME Day 60
27.0 -_+ 2.8
30.4 ±
3.0
25.4 ± 2.5
POA Day 21 Day 60
20.8 --- 4.0 5.7 ± 0.9
21.9 -_+ 2.4 6.4 ± 1.0
30.5 ± 7.6 8.5 ± 0.7
(POA) IN MSG-TREATEDAND CONTROL RATS Group
NE
DA
5-HIAA
Day 21 Control MSG-Lo MSG-Hi
49.7 _+ 7.2 73.1 __- 9.2 56.0 ± 6.7
14.8 ± 3.3 18.1 _+ 3.2 17.6 ± 2.7
ND ND ND
Day 60 Control MSG-Lo MSG°Hi
67.9 ± 5.5 57.7 ± 7.4 60.6 _ 5.0
6.0 ± 0.5 8.4 ± 1.4 7.4 ± 1.4
4.8 ± 0.5 6.2 - 0.5* 6.8 ± 0.5*
All values expressed as pg/Ixg protein ± SEM. N = 7-15 per group; ND = not detectable. *p<0.05 control vs. MSG-treated.
All values expressed as pg/Ixg protein ± SE. N = 8-15 per group.
natal day 21. The MSG-Hi rats also had significantly lower LME DA content than MSG-Lo rats on postnatal day 60 (Fig. 2). There was a significant (p<0.001) age-related increase in LME DA content from postnatal day 21 to 60. DOPAC content was not altered in the LME by M S G treatment. NE content was significantly ( p < 0 . 0 5 ) decreased in MSG-Hi rats when compared to controls on both postnatal day 21 and 60 (Fig. 3). There were no prominent age-related changes in NE content in the LME. No significant changes in NE, DA or DOPAC content in the preoptic area (POA) were found due to M S G treatment (Tables 1 and 2). The serotonin (5-HT) metabolite, 5-hydroxyindoleacetic acid (5-HIAA), was significantly ( p < 0 . 0 5 ) increased in the POA of MSG-treated rats on postnatal day 60 when compared to the controls (Table 2). DA and DOPAC content in the POA on postnatal day 21 were significantly ( p < 0 . 0 5 ) higher in all groups when compared to day 60 (Tables 1 and 2). DA content was determined in the neurointermediate lobe (NIL) and on postnatal day 21 there was no difference between control rats (33.1 m 2.6 pg/~g protein, n = 9) and rats treated with MSG (MSG-Lo, 34.2-+3.8, n = 10; MSG-Hi 26.3---2.3, n = 12). DA content was also unaltered by M S G treatment on postnatal day 60 (control=23.3--- 1.3, n = 16; M S G - L o = 2 3 . 5 -+ 1.5, n = 15; MSG-Hi = 23.6 - 1.2, n = 13). There was a significant (p<0.001)
MEDIAL
age-related decline in NIL DA content. DA and DA metabolites were also measured in tissue punches taken from the striatum as a control tissue since the striatum is not considered a target for the neonatal neurotoxicity of MSG. There was no significant effect of M S G treatment on striatal DA or DA metabolites, although significant (p<0.001) age-related increases in DA and DA metabolites did occur (Table 3). M S G has been reported to damage brainstem structures (28) and alter NE metabolism in the pons-meduUa of mice (12). We measured monoamine content in the pons-medulla of control and MSG-treated rats to examine extra-hypothalamic alterations in monoamine metabolism that could be attributable to neonatal MSG administration. No significant changes were found in tissue stores of NE, 5-HT or 5-HIAA in the pons-medulla of MSGtreated rats except for a reduction in 5-HIAA content in 21-day-old MSG-Hi relative to MSG-Lo rats (Table 4). There was no difference in the body weight of 21-day-old MSG-treated rats (MSG-Lo = 38 -+ 2 g; MSG-Hi = 39 -+ 1 g) when compared to controls (37-+ 2 g). In contrast, body weights were significantly (p<0.01) lower in MSG-treated (MSG-Lo = 146 _+3 g; M S G - H i = 154--+4) rats than controls (177_+5 g) on postnatal day 60. There were no significant effects of M S G treatment on the weights of organs from 21-day-old rats (Table 5). Significant reductions in organ weights were present in MSG-treated rats on postnatal day 60 reflecting the apparent growth stunting. There
TABLE 3
ME
LATERAL ME
50-
[]
CONTROL
]
LOW HIGH
DA, DOPAC AND HOMOVANILL1C ACID (HVA) CONTENT IN STRIATAL PUNCHES
Group
DA
DOPAC
HVA
Day 21 Control MSG-Lo MSG-Hi
54.8 - 2.4 54.0 --_ 5.9 65.0 --+ 11.0
10.5 - 0.7 10.5 ± 0.9 12.0 ± 2.0
ND ND ND
Day 60 Control MSG-Lo MSG-Hi
85.9 + 82.3 ± 89.8 ±
14.8 ± 1.2 14.4 ± 0.9 15.9 _-. 1.2
10.2 ± 0.6 11.4 ± 1.0 10.3 ± 0.4
40-
+~ 30z
20-
10" 21
6O
21
60
DAY
FIG. 3. Effects of MSG treatment on NE content in the MME and LME. *p<0.05 MSG-treated versus control.
4.7 3.6 3.4
All values expressed as pg/l~g protein ___ SEM. ND = not detectable; N = 8-15 per group.
334
DAWSON, SIMPKINS AND W A L L A C E
TABLE 4 MONOAMINE CONTENT IN THE PONS-MEDULLA OF MSG-TREATED AND CONTROL RATS
Group
NE
5-HT
5-HIAA
Day 21 Control MSG-Lo MSG-Hi
643 ± 11 679 ±- 13 646 ± 12
660 _+ 13 677 -+ 14 663 ± 14
784 --- 48 829 --- 52 705 ± 25*
Day 60 Control MSG-Lo MSG-Hi
531 --. 20 508 ± 22 545 ± 23
513 ± 65 457 ± 66 593 --- 73
291 ± 28 272 ± 32 321 ± 28
All values expressed as ng/g wet tissue weight ± SEM. N = 12-16 per group. *p<0.05 MSG-Lo vs. MSG-Hi.
were no obvious dose-dependent effects of M S G treatment on body or organ weights at postnatal day 60. MSG treatment produced significant reductions in the absolute weights of all organs examined except for the spleen (MSG-Lo) and uteri (MSG-Hi). The day of vaginal opening appeared to occur slightly earlier in MSG-treated rats, but there was no statistically significant difference between MSG-treated ( M S G - L o = d a y 34.6___0.5; MSG-
TABLE 5 ORGAN WEIGHTS IN MSG-TREATED RATS
Organ
Controls
Adrenals (rag) Day 21 9.1 Day 60 65.0
MSG-Lo
MSG-Hi
4-_ 0.7 ± 4
9.4 46.0
--- 0.5 --- 2.0
8.7 42.0
_+ 0.4 ± 3.0
Anterior Pituitary (mg) Day 21 ND Day 60 8.1 ± 0.4
6.1
ND +_ 0.4*
5.8
ND - 0.4*
137.0 540.0
~ 8.0 _+ 19.0"
136.0 552.0
± 6.0 ± 26.0*
Heart (mg) Day 21 144.0 Day 60 640.0
+ 10.0 _+ 21.0
Kidney (g) Day 21 Day 60
0.49 +_ 0.04 1.80 ± 0.05
0.49 ± 0.04 1.45 --- 0.04*
0.48 ± 0.01 1.46 _-. 0.05*
Liver (g) Day 21 Day 60
1.49 ± 8.16 ±
1.48 ± 0.10 7.02 -+ 0.31"
1.50 ± 7.23 ±
0.12 0.29
0.05 0.22*
Ovaries (nag) Day 21 12.9 Day 60 139.0
± ±
1.1 5.0
12.4 113.0
--- 0.8 --- 5.0*
13.0 105.0
± 1.1 --- 7.0*
Spleen (mg) Day 21 133.0 Day 60 632.0
± 17.0 ± 51.0
137.0 541.0
± 11.0 - 29.0
124.0 486.0
_+ 6.0 ~- 26.0*
Uteri (mg) Day 21 21.0 Day 60 496.0
± 1.3 ± 24.0
20.0 412.0
± 1.0 ± 29.0*
21.0 458.0
± 0.9 __+ 32.0
ND = Not determined. *p<0.05 Control vs. MSG-treated.
H i = d a y 34.3+--0.6) and control rats (35.3---0.4). Chi-square analysis of the data also failed to show a difference between control and MSG-treated rats in the frequency of early vaginal opening. Examination of the vaginal smears of control and MSG-treated rats also failed to discern any substantial changes in the length or pattern in estrus cycle due to MSG treatment. DISCUSSION
The examination of discrete hypothalamic regions in pre- and postpubertal MSG-treated female rats revealed both age- and dose-related alterations in monoamines. The developmental profile of neurochemical changes present in microdissected hypothalamic areas of MSG-treated rats differs from the alterations in monoamine content present when the whole mediobasal hypothalamus is examined (13). This discrepancy may arise from the exclusion of hypothalamic areas such as the ventromedial nucleus that receive axonal projections from the tyrosine hydroxylase positive neurons of the arcuate nucleus (5). The failure to detect significant depletions in DA in the hypothalamic areas examined in the MSG-Lo rats, despite the pituitary atrophy and changes in organ weights, suggests that events unrelated to direct excitotoxicity may cause organizational changes in the mediobasal hypothalamus that are equally disruptive to normal hypothalamic function. Rats treated with the same dosing regimen as the MSG-Lo rats in this study do show reductions in DA, DOPAC and neuropeptide Y content (13,54) and significant decreases in DA and G A B A uptake in the mediobasal hypothalamus when examined as adults (11). Studies in our laboratory have also confirmed through the use of histological techniques the loss of ANH neurons in MSG-Lo rats (11,13). The timing and dose of the postnatal injections of M S G may not only influence the direct neurotoxicity of MSG (50), but may influence the ontogenic development of mediobasal hypothalamic neuronal populations and the postnatal establishment of synapse formation (8,32). Administration of the excitatory amino acid analog, N-methyl-DL-aspartic acid to immature female rats activates the release of LHRH and advances the onset of puberty (53). Neonatal administration of neurotoxic doses of MSG may also evoke significant acute neurochemical (10) and neuroendocrine responses (39,43) that could result in permanent modification of the anatomical integrity of the mediobasal hypothalamus. Recent evidence points to a direct role for glutamate as a neuronal growth factor (2). Thus, neurons not damaged by MSG may be inappropriately stimulated to initiate growth and differentiation. The obesity and endocrine dysfunction that results from neonatal MSG treatment has been assumed to be due to the direct excitotoxic action of glutamate. The fact that the ontogenesis and migration of dopamine utilizing neurons in the hypothalamus are altered by neonatal MSG suggests that MSG treatment may alter the organization of neural inputs onto target neurons in the mediobasal hypothalamus. The ability of glutamate to act as a neuronal growth factor and influence neuronal differentiation (2) could also result in abnormal connectivity of neural circuits involved in controlling pulsatile hormone release or other integrative hypothalamic functions. Evidence from our laboratory also has demonstrated that the hypothalamic dopamine neurons that survive MSG treatment in mice exhibit neurochemical characteristics that are dissimilar to those normally seen in tuberoinfundibular DA neurons (14). Thus, significant reorganizational, compensatory or diaschisatic changes may occur in the hypothalamus due to both the direct and in direct actions of neonatal MSG administration. The ANH is a primary anatomical locus for MSG-induced neuronal loss, although significant neuronal damage is present in all of the circumventricular organs (28). The anterior portion of the ANH is much more sensitive to the excitotoxic action of M S G than
DEVELOPMENTAL EFFECTS OF MSG
335
is the posterior part of the ANH (50). We have demonstrated that tuberoinfundibular DA neurons are damaged by MSG, whereas tuberohypophyseal DA neurons are relatively spared (11). In the present study, we have again demonstrated NIL DA content was not altered by MSG treatment. Studies of the effects of MSG on immunoreactive tyrosine hydroxylase-containing neurons have demonstrated reductions in their numbers within the ANH, but some tyrosine hydroxylase positive neurons are resistant to the neurotoxic effects of MSG (8, 11, 22). The DOPAC levels of MSG-treated rats were normal even when DA content was reduced so DA turnover rates may be increased in the DA neurons that survive MSG treatment as is the case in MSG-treated mice (14). The ANH is reported to contain at least fifteen different neurotransmitters and neuropeptides (6). The ANH contains neurons with both short axon and long axon efferents and intrahypothalamic projections are extensive (5,6). MSG treatment results in a large loss of estradiol target neurons in the ANH (22). The extensive postnatal estrogen-dependent synaptogenesis that occurs in the ANH and hypothalamus (1, 33, 55) would certainly be disrupted by MSG treatment as would many postsynaptic sites that normally receive input from ANH neurons. The values for the DA content in the MME and LME of the 60-day-old females are in excellent agreement with the findings of Selmanoff (49). The significant reductions in both DA and NE in the LME of MSG-Hi rats suggest that the MSG-induced destruction of the arcuate nucleus disrupts the release of neuropeptides from the median eminence (15,51) by eliminating important modulatory inputs. Catecholamines (DA and NE) are known to be important in regulating the release of a number of hypothalamic neuropeptides and anatomic studies have suggested that in the median eminence direct axon-axonic contacts exist between catecholamine terminals and LHRH terminals (20). No significant changes were seen in the MME content of either DA or NE in MSG-treated rats. This result is in agreement with Daikoku et al. (8) who reported that tyrosine hydroxylase immunoreactive fibers remained almost intact in the medial portion of the median eminence. The lack of significant changes in MME DA or DOPAC content in MSG-treated rats may explain the ability of MSG-treated rats to maintain prolactin levels within normal limits under unstressed conditions. Conversely, the depletion of NE and DA in the LME would appear to contribute to the reproductive dysfunction in MSG-treated rats. Thus, the most profound alterations found in this study due to MSG treatment occur in neurons projecting to the LME. A number of age-related changes in monoamines were observed in this study. In general, DA content in the hypothalamus increased with age, except in the POA. DA and DOPAC content in the POA was 2-3-fold higher on day 21 when compared to day 60. DA-stimulated adenylate cyclase activity and tyrosine hydroxylase activity in the POA (23,27) and DA receptor number in the hypothalamus (19) reach their highest levels around postnatal day 21. Thus, the changes in POA DA metabolism appear important in sexual maturation (56). MSG treatment did not alter DA or DOPAC content in the POA and vaginal opening and early estrus cycles appeared normal. Serotonin metabolism as indexed by 5-HIAA levels did, however, appear increased in postpubertal MSG-treated females. Our results confirm those of Bakke et al. (3) who also reported no significant alterations in the day of
vaginal opening or early estrus cycles. The day of vaginal opening has been reported to be delayed (46) or accelerated (34) by MSG treatment in rats. There is no question that ovarian atrophy results from MSG treatment and estrus cycle and reproductive dysfunction do develop over time in MSG-treated rats (13,40). The changes in postpubertal 5-HIAA, NE or DA could contribute to the late developing reproductive abnormalities in MSG-treated rats. The discrepancies in the early indicators of reproductive dysfunction in MSG-treated rats most likely are attributable to the dose and timing of MSG administration. There were no significant age-related changes in hypothalamic NE content, except in the ANH. Control females exhibited an age-related increase in NE content, but MSG-treated females did not significantly increase their ANH NE content from day 21 values. This effect could be the result of a loss of specific target neurons in the ANH of MSG-treated rats such that the NE innervation of the ANH does not develop properly. Destruction of ANH neurons or alterations in synaptic contacts produced by neonatal MSG treatment may be responsible for the depletion of NE in the LME. We have previously demonstrated significant reduction in NE content in the mediobasal hypothalamus of 60-day-old female MSG-treated rats (13). Johnston et al. (24) reported NE content to be decreased in the ANH and increased in the median eminence of 5-7-month-old ovariectomized MSGtreated rats. MSG treatment can alter NE stores in discrete hypothalamic nuclei which may be important for the expression of certain aspects of the MSG syndrome. In summary, significant age- and dose-related alterations in monoamine content were found in microdissected regions of the hypothalamus in MSG-treated female rats. There was also noted several significant age-related changes in hypothalamic monoamine content, most notably in the POA. The reductions in pituitary and organ weights did not greatly discriminate the two dosing regimens employed, but there were significant differences in the degree of MSG-induced monoamine depletion. This suggests that the loss of nondopaminergic neurons and/or the alterations in postnatal development of the hypothalamus induced by MSG treatment (8,32) are sufficient to produce most of the characteristics of the MSG syndrome in adult rats. Critical periods may exist during the postnatal maturation of the mediobasal hypothalamus when MSG administration will have both profound direct excitotoxic action and indirect reorganizational actions. This may coincide with the increased sensitivity of the neonatal brain to glutamate-induced inositol lipid hydrolysis (41) and/or the late postnatal development of glutamate uptake capacity. The fact that resistance to the excitotoxic actions of systemically administered MSG increases with postnatal development may be related to both the maturation of neuroprotective mechanisms and the postnatal establishment of appropriate neuronal connectivity within the mediobasal hypothalamus. ACKNOWLEDGEMENTS This project was funded in part by a BRSG grant from the College of Pharmacy, University of Florida and a New Faculty Research Support Grant from the Division of Sponsored Research at the University of Florida. The authors wish to express their thanks to Victoria Redd Patterson and Carol Powell for their excellentsecretarial assistance.
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doses of L-monosodium glutamate promote neuronal growth and differentiationin vitro. Dev. Neurosci. 9:228-239; 1987. 3. Bakke, J. L.; Lawrence, N.; Bennett,J.; Robinson,S.; Bowers, C. Y. Late endocrine effects of administeringmonosodiumglutamate to neonatal rats. Neuroendocrinology26:220-228; 1978.
336
4. Bradford, M. M. A rapid sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye-binding. Anal. Biochem. 72:248-254; 1976. 5. Chan-Palay, V.; Zaborszky, L.; Kohler, C.; Goldstein, M.; Palay, S. L. Distribution of tyrosine-hydroxylase-immunoreactive neurons in the hypothalamus of rats. J. Comp. Neurol. 227:467-496; 1984. 6. Chronwall, B. M. Anatomy and physiology of the neuroendocrine accurate nucleus. Peptides 6(Suppl. 2): 1-11; 1985. 7. Clemens, J. A.; Roush, M. E.; Fuller, R. W.; Shaar, C. J. Changes in luteinizing hormone and prolactin control mechanisms produced by glutamate lesions of the arcuate nucleus. Endocrinology 103:13041312; 1978. 8. Daikoku, S.; Kawano, H.; Okamura, Y.; Tokuzen, M.; Nagatsu, I. Ontogenesis of immunoreactive tyrosine hydroxylase-containing neurons in rat hypothalamus. Dev. Brain Res. 28:85-98; 1986. 9. Dawson, R., Jr.; Lorden, J. F. Behavioral and neurochemical effects of neonatal administration of monosodium L-glutamate in mice. J. Comp. Physiol. Psychol. 95:71-84; 1981. 10. Dawson, R. Acute and long lasting neurochemicai effects of monosodium glutamate administration to mice. Neuropharmacology 22: 1417-1419; 1983. 11. Dawson, R., Jr.; Valdes, J. J.; Annau, Z. Tuberohypophyseal and tuberoinfundibular dopamine systems exhibit differential sensitivity to neonatal monosodium glutamate treatment. Pharmacology 31:17-23; 1985. 12. Dawson, R., Jr.; Annau, Z. Neonatal monosodium glutamate administration alters noradrenergic measures in the brainstem of the mouse. Brain Res. Bull. 15:117-121; 1985. 13. Dawson, R., Jr. Developmental and sex-specific effects of low dose neonatal monosodium glutamate administration on mediobasal hypothalamic chemistry. Neuroendocrinology 42:158-166; 1986. 14. Dawson, R.; Callahan, M. F.; Annau, Z. Hypothalamus monoamine metabolism in mice: evaluation of drug challenges and neurotoxic insult. Pharmacology 32:25-37; 1986. 15. DePaolo, L. V.; Negro-Vilar, A. Neonatal monosodium glutamate treatment alters the response of median eminence luteinizing hormone-releasing hormone nerve terminals to potassium and prostaglandin E2. Endocrinology 110:835-841; 1982. 16. Dunnett, C. W. Multiple comparisons. In: McArthur, J. W.; Colton, T., eds. Statistics in endocrinology. Cambridge, MA: MIT Press; 1970:79-103. 17. Estes, K. S.; Simpkins, J. W. Age-related alterations in dopamine and norepinephrine activity within microdissected brain regions of ovariectomized Long Evans rats. Brain Res. 298:209-218; 1984. 18. Greeley, G. H., Jr.; Nicholson, G. F.; Kiser, J. S. A delayed LH/FSH rise after gonadectomy and a delayed serum TSH rise after thyroidectomy in monosodium L-glutamate (MSG)-treated rats. Brain Res. 195:111-122; 1980. 19. Herdon, H. J.; Wilson, C. A. Changes in hypothalamic dopamine D-2 receptors during sexual maturation in male and female rats. Brain Res. 343:151-153; 1985. 20. Ibata, Y.; Tani, N.; Obata, H. L.; Tanaka, M.; Kubo, S.; Fukui, K.; Fujimoto, M.; Kinoshita, H.; Watanabe, K.; Sano, Y. Correlative ontogenetic development of catecholamine- and LHRH-containing nerve endings in the median eminence of the rat. Cell Tissue Res. 216:31-38; 1981. 21. Inkster, S. E.; Clayton, R. N.; Whitehead, S. A. Pituitary receptors for LH-releasing hormone (LHRH) and responsiveness to LHRH in adult female rats after neonatal monosodium L-glutamate treatment. J. Endocrinol. 107:9-13; 1985. 22. Jennes, L.; Stumpf, W. E.; Bisette, G.; Nemeroff, C. B. Monosodium glutamate lesions in rat hypothalamus studied by immunohistochemistry for gonadotxopin releasing hormone, neurotensin, tyrosine hydroxyglase and glutamic acid decarboxylase and by autoradiography for [3H]estradiol. Brain Res. 308:245-253; 1984. 23. Jimenez, A. E.; Meyer, D. C.; Murphy, P. J. Developmental patterns of tyrosine hydroxylase activity in discrete central nervous system regions and serum LH and prolactin in the prepubertal rat. Neuroendocrinology 38:134-138; 1984. 24. Johnston, C. A.; Tesone, M.; Negro-Vilar, A. Steroid-monoamine feedback interactions indiscrete brain regions using as a model the monosodium glutamate (MSG)-lesioned rat. Life Sci. 34:1287-1297; 1984.
DAWSON, SIMPKINS AND WALLACE
25. Kontur, P.; Dawson, R.; Monjan, A. A. Manipulation of mobile phase parameters for the HPLC separation of endogenous monoamines in rat brain tissue. J. Neurosci. Methods 11:5-18; 1984. 26. Koritsanzky, S. Ctyo- and synaptogonesis in the arcuate nucleus of the rat hypothalamus during fetal and early postnatal life. Cell Tissue Res. 200:135-146; 1979. 27. Lamberts, R.; Wuttke, W. Puberty of female rats may in part be explained by decreased hypothalamic dopamine receptor sensitivity. Brain Res. 215:375-381; 1981. 28. Lemkey-Johnston, N.; Reynolds, W. A. Nature and extent of brain lesions in mice related to ingestion of monosodium glutamate: a light and electron microscope study. J. Neuropathol. Exp. Neurol. 33: 74-97; 1974. 29. Loiszou, L. A. The postnatal development of monoamine-containing structures in the hypothalami-hypophyseal system of the albino rat. Z. Zellforsch. 114:234-252; 1971. 30. McNeill, T. H.; Scott, D. E.; Sladek, J. R., Jr. Simultaneous monoamine histofluorescence and neuropeptide immunocytochemistry: I. Localization of catecholamines and gonadotropin-releasing hormone in the rat median eminence. Peptides 1:59-68; 1980. 31. Marani, E.; Rietveld, W. J.; Boon, M. E.; Gerrits, N. M. Fluorescence-displacement from the median eminence toward the arcuate nucleus at puberty. Histochemistry 73:165-169; 1981. 32. Marani, E.; Rietveld, W. J.; Boon, M. E. Monosodium glutamate accelerate migration of hypothalamic perikarya at puberty. Histochemistry 75:145-150; 1982. 33. Matsumoto, A.; Arai, Y. Developmental changes in synaptic formation in the hypothalamic arcuate nucleus of female rats. Cell Tissue Res. 169:143-156; 1976. 34. Matsuzawa, Y.; Yonetani, S.; Takasaki, Y.; Iwata, S.; Sekine, S. Studies on reproductive endocrine function in rats treated with monosodium L-glutamate early in life. Toxicol. Lett. 4:359-371; 1979. 35. Mechanick, J. I.; Cohen-Becker, I. R.; Gregerson, K. A.; Selmanoff, M. Distribution of 3,4-dihydroxyphenylacetic acid (DOPAC) and 3,4-dihydroxyphenylglycol (DOPEG) in microdissected brain structures and the pituitary gland: metabolite changes in the median eminence in response to hyperprolactinemia and suckling. J. Neural Transm. 68:197-215; 1987. 36. Millard, W. J.; Martin, J. B., Jr.; Audet, J.; Sagar, S. M.; Martin, J. B. Evidence that reduced growth hormone secretion observed in monosodium glutamate-treated rats is the result of a deficiency in growth hormone-releasing factor. Endocrinology 110:540-549; 1982. 37. Moore, K. E.; Demarest, K. T. Tuberoinfundibular and tuberohypophyseal dopaminergic neurons. In: Ganong, W. F.; Martin, L., eds. Frontiers in neuroendocrinology, vol. 7. New York: Raven Press; 1982:161-190. 38. Nemeroff, C. B.; Konkol, R. J.; Bissette, G.; Youngblood, W.; Martin, J. B.; Brazeau, P.; Rone, M. S.; Prange, A. J., Jr.; Breese, G, R.; Kiser, J. S. Analysis of the disruption in hypothalamicpituitary regulation in rats treated neonatally with monosodium L-glutamate (MSG): Evidence for the involvement of tuberoinfundibular cholinergic and dopaminergic systems in neuroendocrine regulation. Endocrinology 101:613-622; 1977. 39. Nemeroff, C. B.; Bissette, G.; Greeley, G. H.; Mailman, R. B.; Martin, J. B.; Brazeau, P.; Kizer, J, S. Effects of acute administration of monosodium-L-glutamate (MSG), atropine or haloperidol on anterior pituitary hormone secretion in the rat. Brain Res. 156:198-201; 1978. 40. Nemeroff, C. B.; Lamartiniere, C. A.; Mason, G. A.; Squibb, R. E.; Hong, J. S.; Bondy, S. C. Marked reduction in gonadal steroid hormone levels in rats treated neonatally wtih monosodium Lglutamate: further evidence for disruption of hypothalamic-pituitarygonadal axis regulation. Neuroendocrinology 33:265-267; 1981. 41. Nicoletti, F.; Iadarola, M. J.; Wroblewski, J. T.; Costa, E. Excitatory amino acid recognition sites coupled with inositol phospholipid metabolism: developmental changes and interaction with al-adrenoceptors. Proc. Natl. Acad. Sci. USA 83:1931-1935; 1986. 42. Nikoletseas, M. Obesity in exercising, hypophagic rats treated with monosodium glutamate. Physiol. Behav. 19:767-773; 1977. 43. Olney, J. W.; Cicero, T. J.; Meyer, E. R.; DeGubareff, T. Acute glutamate-induced elevations in serum testosterone and luteinizing hormone. Brain Res. 112:420--424; 1976.
DEVELOPMENTAL EFFECTS OF MSG
44. Palkovits, M. Isolated removal of hypothalamic or other brain nuclei of the rat. Brain Res 59:449--450; 1973. 45. Pearce, I. A.; Cambray-Deakin, M. A.; Burgoyne, R. D. Glutamate acting on NMDA receptors stimulated neurite outgrowth from cerebellar granule cells. FEBS Lett. 223:143-147; 1987. 46. Rodriguez-Sierra, J. F.; Sridaran, R.; Blake, C. A. Monosodium glutamate disruption of behavioral and endocrine function in the female rat. Neuroendocrinology 31:228-235; 1980. 47. Rodriguez-Sierra, J. F.; Blaustein, J. D,; Blake, C. A.; Clough, R. W.; Elias, K. A. A decrease of cytosol estrogen receptors in the hypothalamus as a result of treatment of neonatal rats with glutamate. Exp. Brain Res. 48:272-278; 1982. 48. Rose, P. A.; Weick, R. F. Evidence for reorganization of neuroendocrine centers regulating pulsatile LH secretion in rats receiving neonatal monosodium-L-glutamate treatment. J. Endocrinol. 113: 261-269; 1987. 49. Selmanoff, M. The lateral and medial median eminence: distribution of dopamine norepinephrine, and luteinizing hormone-releasing hormone and the effect of prolactin on catecholamine turnover. Endocrinology 108:1716--1722; 1981. 50. Seress, L. Divergent effects of acute and chronic monosodium
337
51. 52.
53. 54.
55. 56.
L-glutamate treatment on the anterior and posterior parts of the arcuate nucleus. Neuroscience 7:2207-2216; 1982. Spinedi, E.; Johnston, C.; Negro-Vilar, A. Increased responsiveness of the hypothalamic-pituitary axis after neurotoxin-induced hypothalamic denervation. Endocrinology 115:267-272; 1984. Sridaran, R.; Rodriguez-Sierra, J. F.; Blake, C. A. Effects of hypothalamic arcuate nucleus lesions on pulsatile luteinizing hormone concentration in overectomized rats (41231). Proc. Soc. Exp. Biol. Med. 168:38--44; 1981. Urbanski, H. F.; Ojeda, S. R. Activation of luteinizing hnrmonereleasing hormone release advances the onset of female puberty. Neuroendocrinology 46:273-276; 1987. Wallace, D. R.; Gabriel, S. M.; Dawson, R. Monosodium glutamateinduced alterations in feeding behavior, blood pressure and neuropeptide Y levels. Soc. Neurosci. Abstr. 12:1454; 1986. Walsh, R. J.; Brawer, J. R.; Naftolin, F. Early postnatal development of the arcuate nucleus in normal and sexually reversed male and female rats. J. Anat. 135:733-744; 1982. Wuttke, W.; Honma, K.; Lamberts, R.; Hohn, K. G. The role of monoamines in female puberty. Fed. Proc. 39:2378-2383; 1980.
0892-0362/89 $3.00 + .00
Age- and Dose-Dependent Effects of Neonatal Monosodium Glutamate (MSG) Administration to Female Rats R. D A W S O N , J R . , J. W. S I M P K I N S A N D D. R. W A L L A C E
Department of Pharmacodynamics, University of Florida, College of Pharmacy JHMHC, Box J-487, Gainesville, FL 32610 R e c e i v e d 22 July 1988
DAWSON, R., JR., J. W. SIMPKINS AND D. R. WALLACE. Age- and dose-dependent effects of neonatal monosodium glutamate (MSG) administration to female rats. NEUROTOXICOL TERATOL 11(4) 331-337, 1989.--The age- and dose-dependent effects of neonatal MSG were evaluated in pre- and postpubertal female rats. The neurotoxic action of MSG was assessed by examining monoamine content in microdissected regions of the mediobasal hypothalamus. MSG was administered at a dose of 4 mg/g on postnatal days 2 and 4 (MSG-Lo) or on postnatal days 2, 4, 6 and 8 (MSG-Hi). MSG-Hi treatment significantly reduced dopamine (DA) content in the arcuate nucleus (ANH) and lateral median eminence (LME) on postnatal day 21 when compared to NaCl-injected controls. DA content relative to controls was not altered in the ANH or LME postnatal or postnatal day 60 in MSG-Hi, however, norepinephrine (NE) was significantly (p<0.05) decreased on both postnatal day 21 and 60 in the LME. MSG-Lo treatment significantly (p<0.05) reduced ANH NE content on postnatal day 60 compared to controls. Both MSG-Hi and MSG-Lo treatment increased 5-hydroxyindoleacetic acid content in the preoptic area (POA) on postnatal day 60 relative to the controls. Developmental changes independent of MSG treatment were noted in the hypothalamus. DA and 3,4-dihydroxyphenylacetic acid (DOPAC) content in the POA were 2-3-fold higher on postnatal day 21 compared to postnatal day 60. In contrast, DA content increased with age in the ANH, LME and medial ME. NE content in the ANH increased as a function of age in controls, but not in MSG-treated rats, The effects of MSG treatment on the postnatal development and maturation of neurons in the mediobasal hypothalamus were discussed in relation to the direct neurotoxicity of MSG. Monosodium glutamate Excitotoxin
Arcuate nucleus
Developmental neurochemistry
Dopamine
Median eminence
The DA neurons that project to the medial portion of the median eminence appear distinct in functional characteristics from the DA neurons that project to the lateral median eminence (35,49). Median eminence DA content has been reported to be decreased (38) or unaltered (24) by MSG treatment. Basal prolactin levels are usually unaltered by MSG treatment (7,38), although hyperprolactinemia has been reported (21,47). LH regulation is generally reported to be disrupted by MSG treatment (7, 18, 24, 52) and gonadal atrophy is a hallmark of the MSG syndrome (11, 40, 46). Thus, there appears to be a greater impact of MSG treatment on neuronal systems projecting to the lateral median eminence (LME), which include LH releasing hormone (LHRH) containing fibers (20,30), in comparison to the medial median eminence (MME) which appears important in mediating the tonic dopaminergic inhibition of prolactin release (37). The aim of the present study was to examine the effects of MSG treatment on the postnatal neurochemical development of discrete mediobasal hypothalamic regions including the LME and MME at pre- and postpubertal time points. A second aim was to examine the dose-response relationship of MSG treatment by employing two different dosing regimens, since the timing of injections, as well as the dose, may alter the functional consequences of MSG treatment. The pronounced ontogenic changes
THE developmental profile of mediobasal hypothalamic monoamine metabolism has previously been reported to be altered by neonatal MSG treatment (13). The arcuate nucleus of the hypothalamus (ANH) is a major target for the neurotoxic action of MSG and the neuronal loss that accompanies MSG treatment results in numerous neuroendocrine deficits involving metabolic, reproductive and growth disturbances (3, 9, 34, 36, 38, 46). Previous studies indicate that at birth the ANH is in an immature state of histogenesis and undergoes an extensive postnatal development (8, 26, 31, 32). The ANH undergoes progressive structural maturation during the sexual differentiation of the hypothalamus (1, 20, 33, 56). Significant organizational and synaptogenic events occur during the ontogenetic development of the ANH as a prelude to full development of a functional hypothalamic-pituitary axis. MSG treatment appears to alter the ontogenesis of ANH dopamine (DA) neurons (8,32) and evokes reorganization of neuroendocrine centers regulating pulsatile luteinizing hormone (LH) secretion (48). MSG may, therefore, exert its effects both by direct excitotoxic mechanisms and also indirectly by altering the postnatal ontogenic development of neuronal and synaptic elements in the mediobasal hypothalamus. Anatomical and neurochemical data suggest that the DA neurons of the ANH exhibit functional heterogeneity (8, 11, 49).
331
332
DAWSON, SIMPKINS AND WALLACE
occurring as a result of the postnatal and sexual maturation of the ANH make this region an especially vulnerable target for the neurotoxic actions of MSG.
ARCUATE NUCLEUS 50.
I---] CONTROL"
I
40.
METHOD
+J 30-
~
HIGH
")(" ==
~.~.
Animals
,
20-
Timed pregnant Sprague-Dawley rats were obtained from Charles River Breeding Laboratory (Wilmington, MA) and their offspring served as subjects in this experiment. MSG (400 mg/ml in distilled water) was administered subcutaneously at a dose of 4 mg/g on postnatal days 2 and 4 (MSG-Lo) or postnatal days 2, 4, 6 and 8 (MSG-Hi). Littermates were injected subcutaneously with 2.0% sodium chloride as a hypertonic control. All rat pups were randomly assigned to dams after the injection procedure on postnatal day 2 and were cross-fostered until weaning on postnatal day 21. One group of female MSG-treated and control rats were killed by decapitation on postnatal day 21 and the brains were rapidly removed and placed on dry ice. The frozen brains were stored at - 8 0 ° C until processing for microdissection by a modification (17) of the method of Palkovits (44). The remaining female rats were group housed according to treatment condition and checked daily for the time of vaginal opening. Vaginal smears were taken daily by lavage after vaginal opening until postnatal day 60. On postnatal day 60, the rats were killed by decapitation and the brains were processed as described above. Body weights and organ weights were determined in both 21- and 60-day-old rats. All rats were maintained on a 12-hour light-dark cycle with food and water available ad lib.
Neurochemistry Tissue punches were collected in 1.5-ml microcentrifuge tubes containing 40 p~l of 0.1 M perchloric acid which contained 100 mg/1 Na2EDTA and 1.0 mM glutathione. Samples were homogenized by sonication (Heat Systems-Ultrasonics, Plainview, NY) and centrifuged for 4 minutes in a Beckman microfuge (Palo Alto, CA). The supernatant was diluted 1:1 with mobile phase and analyzed for monoamine and metabolite content using high performance liquid chromatography with electrochemical detection (25). The limits of detection for monoamine and metabolite content were approximately 20 pg/20 txl injection at a detector sensitivity of 1.0 nA. Protein content of the tissue pellet was determined using the method of Bradford (4). Data were subjected to two-way analysis of variance (ANOVA) to determine the overall effect of age and MSG treatment and any age x dose interactions. Individual planned comparisons were performed using the least significant difference (LSD) test (16).
t.u z 10-
2420. ~16+1
~
4-
21
60 DAY
FIG. 1. DA and NE content in the ANH of 21- and 60-day-old female MSG-treated and control rats. *p<0.05 MSG-treated versus control; Op<0.05 MSG-Hi versus MSG-Lo.
neonatal MSG treatment on postnatal day 60. There was an overall significant (p<0.001) age-related increase in MME DA content. The DA metabolite, 3,4-dihydroxyphenylacetic acid (DOPAC), was not detectable in 21-day-old rats, but was detectable in the MME at postnatal dz.y 60 (Table 1). No differences in MME DOPAC were found between MSG-treated and control rats. There were no age- or treatment-related alterations in MME norepinephrine (NE) (Fig. 3). There were significant (/7<0.01) age and treatment effects on DA content in the LME (Fig. 2). DA content in MSG-Hi rats was significantly (p<0.05) lower than controls or MSG-Lo on post-
MEDIAL ME
LATERAL ME
320. 280
RESULTS DA content in the ANH was significantly decreased in MSG-Hi rats on postnatal day 21 when compared to control or MSG-Lo rats (Fig. 1). There was a significant (p<0.001) effect of age on DA content in the ANH, although only the MSG-Hi rats exhibited a significant increase in ANH DA content from postnatal day 21 to 60. MSG treatment significantly (p<0.05) decreased ANH NE content (Fig. 1). No significant changes in ANH NE were present on postnatal day 21, but the significant (p<0.001) age-related increase in ANH NE was attenuated in MSG-treated rats. MSG-Lo rats had significantly (p<0.05) lower ANH NE content than controls on postnatal day 60 (Fig. 1). There was no effect of MSG treatment on MME DA content (Fig. 2). The high variability in the control group on day 21 may have obscured a real depletion in MME DA in the MSG-Hi group. MME DA was not altered by
12, .
]
CONTROL
]
LOW
~ 1 HIGH
240
ram200
~ o.
El
160.
.< 120a
El
804021
60
21
60
DAY
FIG. 2. DA content m the MME and LME of MSG-treated and control rats. *p<0.05 MSG-treated vs. control; Op<0.05 MSG-Hi vs. MSG-Lo.
DEVELOPMENTAL EFFECTS OF M S G
333
TABLE I
TABLE 2
DOPAC LEVELS IN THE HYPOTHALAMUS OF MSG-TREATED AND CONTROL RATS
MONOAMINE AND METABOLITE CONTENT IN THE PREOPTIC AREA
Region
Control
MSG-Lo
AN Day 21 Day 60
9.4 ± 11.9 ±
MME Day 60
43.4 ± 10.0
1.5 1.5
9.5 ± 8.8 ±
MSG-Hi
1.6 0.7
10.5 ± 0.5 10.0 ± 0.9
44.5 ± 10.5
49.7 ± 6.66
LME Day 60
27.0 -_+ 2.8
30.4 ±
3.0
25.4 ± 2.5
POA Day 21 Day 60
20.8 --- 4.0 5.7 ± 0.9
21.9 -_+ 2.4 6.4 ± 1.0
30.5 ± 7.6 8.5 ± 0.7
(POA) IN MSG-TREATEDAND CONTROL RATS Group
NE
DA
5-HIAA
Day 21 Control MSG-Lo MSG-Hi
49.7 _+ 7.2 73.1 __- 9.2 56.0 ± 6.7
14.8 ± 3.3 18.1 _+ 3.2 17.6 ± 2.7
ND ND ND
Day 60 Control MSG-Lo MSG°Hi
67.9 ± 5.5 57.7 ± 7.4 60.6 _ 5.0
6.0 ± 0.5 8.4 ± 1.4 7.4 ± 1.4
4.8 ± 0.5 6.2 - 0.5* 6.8 ± 0.5*
All values expressed as pg/Ixg protein ± SEM. N = 7-15 per group; ND = not detectable. *p<0.05 control vs. MSG-treated.
All values expressed as pg/Ixg protein ± SE. N = 8-15 per group.
natal day 21. The MSG-Hi rats also had significantly lower LME DA content than MSG-Lo rats on postnatal day 60 (Fig. 2). There was a significant (p<0.001) age-related increase in LME DA content from postnatal day 21 to 60. DOPAC content was not altered in the LME by M S G treatment. NE content was significantly ( p < 0 . 0 5 ) decreased in MSG-Hi rats when compared to controls on both postnatal day 21 and 60 (Fig. 3). There were no prominent age-related changes in NE content in the LME. No significant changes in NE, DA or DOPAC content in the preoptic area (POA) were found due to M S G treatment (Tables 1 and 2). The serotonin (5-HT) metabolite, 5-hydroxyindoleacetic acid (5-HIAA), was significantly ( p < 0 . 0 5 ) increased in the POA of MSG-treated rats on postnatal day 60 when compared to the controls (Table 2). DA and DOPAC content in the POA on postnatal day 21 were significantly ( p < 0 . 0 5 ) higher in all groups when compared to day 60 (Tables 1 and 2). DA content was determined in the neurointermediate lobe (NIL) and on postnatal day 21 there was no difference between control rats (33.1 m 2.6 pg/~g protein, n = 9) and rats treated with MSG (MSG-Lo, 34.2-+3.8, n = 10; MSG-Hi 26.3---2.3, n = 12). DA content was also unaltered by M S G treatment on postnatal day 60 (control=23.3--- 1.3, n = 16; M S G - L o = 2 3 . 5 -+ 1.5, n = 15; MSG-Hi = 23.6 - 1.2, n = 13). There was a significant (p<0.001)
MEDIAL
age-related decline in NIL DA content. DA and DA metabolites were also measured in tissue punches taken from the striatum as a control tissue since the striatum is not considered a target for the neonatal neurotoxicity of MSG. There was no significant effect of M S G treatment on striatal DA or DA metabolites, although significant (p<0.001) age-related increases in DA and DA metabolites did occur (Table 3). M S G has been reported to damage brainstem structures (28) and alter NE metabolism in the pons-meduUa of mice (12). We measured monoamine content in the pons-medulla of control and MSG-treated rats to examine extra-hypothalamic alterations in monoamine metabolism that could be attributable to neonatal MSG administration. No significant changes were found in tissue stores of NE, 5-HT or 5-HIAA in the pons-medulla of MSGtreated rats except for a reduction in 5-HIAA content in 21-day-old MSG-Hi relative to MSG-Lo rats (Table 4). There was no difference in the body weight of 21-day-old MSG-treated rats (MSG-Lo = 38 -+ 2 g; MSG-Hi = 39 -+ 1 g) when compared to controls (37-+ 2 g). In contrast, body weights were significantly (p<0.01) lower in MSG-treated (MSG-Lo = 146 _+3 g; M S G - H i = 154--+4) rats than controls (177_+5 g) on postnatal day 60. There were no significant effects of M S G treatment on the weights of organs from 21-day-old rats (Table 5). Significant reductions in organ weights were present in MSG-treated rats on postnatal day 60 reflecting the apparent growth stunting. There
TABLE 3
ME
LATERAL ME
50-
[]
CONTROL
]
LOW HIGH
DA, DOPAC AND HOMOVANILL1C ACID (HVA) CONTENT IN STRIATAL PUNCHES
Group
DA
DOPAC
HVA
Day 21 Control MSG-Lo MSG-Hi
54.8 - 2.4 54.0 --_ 5.9 65.0 --+ 11.0
10.5 - 0.7 10.5 ± 0.9 12.0 ± 2.0
ND ND ND
Day 60 Control MSG-Lo MSG-Hi
85.9 + 82.3 ± 89.8 ±
14.8 ± 1.2 14.4 ± 0.9 15.9 _-. 1.2
10.2 ± 0.6 11.4 ± 1.0 10.3 ± 0.4
40-
+~ 30z
20-
10" 21
6O
21
60
DAY
FIG. 3. Effects of MSG treatment on NE content in the MME and LME. *p<0.05 MSG-treated versus control.
4.7 3.6 3.4
All values expressed as pg/l~g protein ___ SEM. ND = not detectable; N = 8-15 per group.
334
DAWSON, SIMPKINS AND W A L L A C E
TABLE 4 MONOAMINE CONTENT IN THE PONS-MEDULLA OF MSG-TREATED AND CONTROL RATS
Group
NE
5-HT
5-HIAA
Day 21 Control MSG-Lo MSG-Hi
643 ± 11 679 ±- 13 646 ± 12
660 _+ 13 677 -+ 14 663 ± 14
784 --- 48 829 --- 52 705 ± 25*
Day 60 Control MSG-Lo MSG-Hi
531 --. 20 508 ± 22 545 ± 23
513 ± 65 457 ± 66 593 --- 73
291 ± 28 272 ± 32 321 ± 28
All values expressed as ng/g wet tissue weight ± SEM. N = 12-16 per group. *p<0.05 MSG-Lo vs. MSG-Hi.
were no obvious dose-dependent effects of M S G treatment on body or organ weights at postnatal day 60. MSG treatment produced significant reductions in the absolute weights of all organs examined except for the spleen (MSG-Lo) and uteri (MSG-Hi). The day of vaginal opening appeared to occur slightly earlier in MSG-treated rats, but there was no statistically significant difference between MSG-treated ( M S G - L o = d a y 34.6___0.5; MSG-
TABLE 5 ORGAN WEIGHTS IN MSG-TREATED RATS
Organ
Controls
Adrenals (rag) Day 21 9.1 Day 60 65.0
MSG-Lo
MSG-Hi
4-_ 0.7 ± 4
9.4 46.0
--- 0.5 --- 2.0
8.7 42.0
_+ 0.4 ± 3.0
Anterior Pituitary (mg) Day 21 ND Day 60 8.1 ± 0.4
6.1
ND +_ 0.4*
5.8
ND - 0.4*
137.0 540.0
~ 8.0 _+ 19.0"
136.0 552.0
± 6.0 ± 26.0*
Heart (mg) Day 21 144.0 Day 60 640.0
+ 10.0 _+ 21.0
Kidney (g) Day 21 Day 60
0.49 +_ 0.04 1.80 ± 0.05
0.49 ± 0.04 1.45 --- 0.04*
0.48 ± 0.01 1.46 _-. 0.05*
Liver (g) Day 21 Day 60
1.49 ± 8.16 ±
1.48 ± 0.10 7.02 -+ 0.31"
1.50 ± 7.23 ±
0.12 0.29
0.05 0.22*
Ovaries (nag) Day 21 12.9 Day 60 139.0
± ±
1.1 5.0
12.4 113.0
--- 0.8 --- 5.0*
13.0 105.0
± 1.1 --- 7.0*
Spleen (mg) Day 21 133.0 Day 60 632.0
± 17.0 ± 51.0
137.0 541.0
± 11.0 - 29.0
124.0 486.0
_+ 6.0 ~- 26.0*
Uteri (mg) Day 21 21.0 Day 60 496.0
± 1.3 ± 24.0
20.0 412.0
± 1.0 ± 29.0*
21.0 458.0
± 0.9 __+ 32.0
ND = Not determined. *p<0.05 Control vs. MSG-treated.
H i = d a y 34.3+--0.6) and control rats (35.3---0.4). Chi-square analysis of the data also failed to show a difference between control and MSG-treated rats in the frequency of early vaginal opening. Examination of the vaginal smears of control and MSG-treated rats also failed to discern any substantial changes in the length or pattern in estrus cycle due to MSG treatment. DISCUSSION
The examination of discrete hypothalamic regions in pre- and postpubertal MSG-treated female rats revealed both age- and dose-related alterations in monoamines. The developmental profile of neurochemical changes present in microdissected hypothalamic areas of MSG-treated rats differs from the alterations in monoamine content present when the whole mediobasal hypothalamus is examined (13). This discrepancy may arise from the exclusion of hypothalamic areas such as the ventromedial nucleus that receive axonal projections from the tyrosine hydroxylase positive neurons of the arcuate nucleus (5). The failure to detect significant depletions in DA in the hypothalamic areas examined in the MSG-Lo rats, despite the pituitary atrophy and changes in organ weights, suggests that events unrelated to direct excitotoxicity may cause organizational changes in the mediobasal hypothalamus that are equally disruptive to normal hypothalamic function. Rats treated with the same dosing regimen as the MSG-Lo rats in this study do show reductions in DA, DOPAC and neuropeptide Y content (13,54) and significant decreases in DA and G A B A uptake in the mediobasal hypothalamus when examined as adults (11). Studies in our laboratory have also confirmed through the use of histological techniques the loss of ANH neurons in MSG-Lo rats (11,13). The timing and dose of the postnatal injections of M S G may not only influence the direct neurotoxicity of MSG (50), but may influence the ontogenic development of mediobasal hypothalamic neuronal populations and the postnatal establishment of synapse formation (8,32). Administration of the excitatory amino acid analog, N-methyl-DL-aspartic acid to immature female rats activates the release of LHRH and advances the onset of puberty (53). Neonatal administration of neurotoxic doses of MSG may also evoke significant acute neurochemical (10) and neuroendocrine responses (39,43) that could result in permanent modification of the anatomical integrity of the mediobasal hypothalamus. Recent evidence points to a direct role for glutamate as a neuronal growth factor (2). Thus, neurons not damaged by MSG may be inappropriately stimulated to initiate growth and differentiation. The obesity and endocrine dysfunction that results from neonatal MSG treatment has been assumed to be due to the direct excitotoxic action of glutamate. The fact that the ontogenesis and migration of dopamine utilizing neurons in the hypothalamus are altered by neonatal MSG suggests that MSG treatment may alter the organization of neural inputs onto target neurons in the mediobasal hypothalamus. The ability of glutamate to act as a neuronal growth factor and influence neuronal differentiation (2) could also result in abnormal connectivity of neural circuits involved in controlling pulsatile hormone release or other integrative hypothalamic functions. Evidence from our laboratory also has demonstrated that the hypothalamic dopamine neurons that survive MSG treatment in mice exhibit neurochemical characteristics that are dissimilar to those normally seen in tuberoinfundibular DA neurons (14). Thus, significant reorganizational, compensatory or diaschisatic changes may occur in the hypothalamus due to both the direct and in direct actions of neonatal MSG administration. The ANH is a primary anatomical locus for MSG-induced neuronal loss, although significant neuronal damage is present in all of the circumventricular organs (28). The anterior portion of the ANH is much more sensitive to the excitotoxic action of M S G than
DEVELOPMENTAL EFFECTS OF MSG
335
is the posterior part of the ANH (50). We have demonstrated that tuberoinfundibular DA neurons are damaged by MSG, whereas tuberohypophyseal DA neurons are relatively spared (11). In the present study, we have again demonstrated NIL DA content was not altered by MSG treatment. Studies of the effects of MSG on immunoreactive tyrosine hydroxylase-containing neurons have demonstrated reductions in their numbers within the ANH, but some tyrosine hydroxylase positive neurons are resistant to the neurotoxic effects of MSG (8, 11, 22). The DOPAC levels of MSG-treated rats were normal even when DA content was reduced so DA turnover rates may be increased in the DA neurons that survive MSG treatment as is the case in MSG-treated mice (14). The ANH is reported to contain at least fifteen different neurotransmitters and neuropeptides (6). The ANH contains neurons with both short axon and long axon efferents and intrahypothalamic projections are extensive (5,6). MSG treatment results in a large loss of estradiol target neurons in the ANH (22). The extensive postnatal estrogen-dependent synaptogenesis that occurs in the ANH and hypothalamus (1, 33, 55) would certainly be disrupted by MSG treatment as would many postsynaptic sites that normally receive input from ANH neurons. The values for the DA content in the MME and LME of the 60-day-old females are in excellent agreement with the findings of Selmanoff (49). The significant reductions in both DA and NE in the LME of MSG-Hi rats suggest that the MSG-induced destruction of the arcuate nucleus disrupts the release of neuropeptides from the median eminence (15,51) by eliminating important modulatory inputs. Catecholamines (DA and NE) are known to be important in regulating the release of a number of hypothalamic neuropeptides and anatomic studies have suggested that in the median eminence direct axon-axonic contacts exist between catecholamine terminals and LHRH terminals (20). No significant changes were seen in the MME content of either DA or NE in MSG-treated rats. This result is in agreement with Daikoku et al. (8) who reported that tyrosine hydroxylase immunoreactive fibers remained almost intact in the medial portion of the median eminence. The lack of significant changes in MME DA or DOPAC content in MSG-treated rats may explain the ability of MSG-treated rats to maintain prolactin levels within normal limits under unstressed conditions. Conversely, the depletion of NE and DA in the LME would appear to contribute to the reproductive dysfunction in MSG-treated rats. Thus, the most profound alterations found in this study due to MSG treatment occur in neurons projecting to the LME. A number of age-related changes in monoamines were observed in this study. In general, DA content in the hypothalamus increased with age, except in the POA. DA and DOPAC content in the POA was 2-3-fold higher on day 21 when compared to day 60. DA-stimulated adenylate cyclase activity and tyrosine hydroxylase activity in the POA (23,27) and DA receptor number in the hypothalamus (19) reach their highest levels around postnatal day 21. Thus, the changes in POA DA metabolism appear important in sexual maturation (56). MSG treatment did not alter DA or DOPAC content in the POA and vaginal opening and early estrus cycles appeared normal. Serotonin metabolism as indexed by 5-HIAA levels did, however, appear increased in postpubertal MSG-treated females. Our results confirm those of Bakke et al. (3) who also reported no significant alterations in the day of
vaginal opening or early estrus cycles. The day of vaginal opening has been reported to be delayed (46) or accelerated (34) by MSG treatment in rats. There is no question that ovarian atrophy results from MSG treatment and estrus cycle and reproductive dysfunction do develop over time in MSG-treated rats (13,40). The changes in postpubertal 5-HIAA, NE or DA could contribute to the late developing reproductive abnormalities in MSG-treated rats. The discrepancies in the early indicators of reproductive dysfunction in MSG-treated rats most likely are attributable to the dose and timing of MSG administration. There were no significant age-related changes in hypothalamic NE content, except in the ANH. Control females exhibited an age-related increase in NE content, but MSG-treated females did not significantly increase their ANH NE content from day 21 values. This effect could be the result of a loss of specific target neurons in the ANH of MSG-treated rats such that the NE innervation of the ANH does not develop properly. Destruction of ANH neurons or alterations in synaptic contacts produced by neonatal MSG treatment may be responsible for the depletion of NE in the LME. We have previously demonstrated significant reduction in NE content in the mediobasal hypothalamus of 60-day-old female MSG-treated rats (13). Johnston et al. (24) reported NE content to be decreased in the ANH and increased in the median eminence of 5-7-month-old ovariectomized MSGtreated rats. MSG treatment can alter NE stores in discrete hypothalamic nuclei which may be important for the expression of certain aspects of the MSG syndrome. In summary, significant age- and dose-related alterations in monoamine content were found in microdissected regions of the hypothalamus in MSG-treated female rats. There was also noted several significant age-related changes in hypothalamic monoamine content, most notably in the POA. The reductions in pituitary and organ weights did not greatly discriminate the two dosing regimens employed, but there were significant differences in the degree of MSG-induced monoamine depletion. This suggests that the loss of nondopaminergic neurons and/or the alterations in postnatal development of the hypothalamus induced by MSG treatment (8,32) are sufficient to produce most of the characteristics of the MSG syndrome in adult rats. Critical periods may exist during the postnatal maturation of the mediobasal hypothalamus when MSG administration will have both profound direct excitotoxic action and indirect reorganizational actions. This may coincide with the increased sensitivity of the neonatal brain to glutamate-induced inositol lipid hydrolysis (41) and/or the late postnatal development of glutamate uptake capacity. The fact that resistance to the excitotoxic actions of systemically administered MSG increases with postnatal development may be related to both the maturation of neuroprotective mechanisms and the postnatal establishment of appropriate neuronal connectivity within the mediobasal hypothalamus. ACKNOWLEDGEMENTS This project was funded in part by a BRSG grant from the College of Pharmacy, University of Florida and a New Faculty Research Support Grant from the Division of Sponsored Research at the University of Florida. The authors wish to express their thanks to Victoria Redd Patterson and Carol Powell for their excellentsecretarial assistance.
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