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Effects of monosodiuml -glutamate administration on neuropeptide Y-containing neurons in the rat hypothalamus
Hram I{c.scarch, 369 ( l¢),~f~i.:,~ ~)I) ) I',cxw)
388
BRE 21~54
Effects of monosodium L-glutamate administration on neuropeptide Y-containing neurons in the rat hypothalamus L. KERKI~RIAN and G. PELLETIER MRC Group in Molecular Endocrinology, Le Centre Hospitalier de I'UniversitO Laval, Quebec G I V 4G2, Que. (Canada)
(Accepted November 26th, 1985) Key words." L-glutamate - - neuropeptide Y - - hypothalamus
Immunocytochemical localization of neuropeptide Y (NPY) was performed in the hypothalamus of rats of which the arcuate nucleus had been destroyed with monosodium L-glutamate in the neonatal period. The treatment produced a disappearance of most of the NPY cell bodies normally found in the arcuate nucleus. The concentration of fibers was decreased in the paraventricular nucleus, but not in the other hypothalamic nuclei. The treatment also induced the appearance of a large number of immunoreactive cell bodies in the paraventricular nucleus. These results strongly suggest that arcuate NPY neurons are projecting to the paraventricular nucleus and that the arcuate nucleus probably exerts some inhibitory tonic influence on NPY paraventricular neurons.
N e u r o p e p t i d e Y (NPY) is a 36-amino acid residue peptide which has been characterized from porcine brain by T a t e m o t o ~ and T a t e m o t o et al. x2. This peptide has structural analogy with the gut peptide YY (PYY) and pancreatic polypeptide (PP). Recently, Allen et al. 1 using antibodies to porcine N P Y have clearly d e m o n s t r a t e d that PP-like immunoreactivity, found in neurons of rat brain, was due to cross-reactivity of antibodies to PP with NPY. In the rat and man, NPY has been localized by immunohistochemistry in several brain areas. In the hypothalamus, numerous NPY-containing neuronal cell bodies were observed in the arcuate nucleus, whereas high concentration of immunoreaction fibers have been detected in the suprachiasmatic and paraventricular nuclei 1,7.8. A m o d e r a t e l y dense network of positive fibers was also observed in the arcuate nucleus and inner zones of the m e d i a n eminence. The origin of the NPY fibers found in nuclei of the hypothalami has not been extensively studied. Very recently, Bai et ai.2 have shown that electrical lesion in the area of the arcuate nucleus p r o d u c e d a decrease of NPY fibers ipsilaterally in the paraventricular and dorsal lateral nuclei of the rat. In o r d e r to evaluate the pro-
jection of the arcuate N P Y neurons, we p r o c e e d e d to an immunohistochemical localization of NPY after treatment with m o n o s o d i u m L-glutamate ( M S G ) which selectively destroys the arcuate nucleus 3,4.~. Seven female rats received 2.0 mg/g b. wt. on the 2nd and 4th days of life and 4.0 mg/g b. wt. on the 6th, 8th and 10th days. As control, a group of 6 female rats were similarly treated with the vehicle (saline). They were used for immunohistochemistry at 3 months of age. Three ammals p e r group were injected with colchicine (25/~g) into the left lateral ventricle 48 h before fixation. All the animals were fixed by a vascular perfusion of a solution of 4 % paraformaldehyde in 0.1 M p h o s p h a t e buffer (pH 7.4). The brains were then dissected out and i m m e r s e d in the same fixative for 18 h. Consecutive coronal sections (30/~m) were cut with the V i b r a t o m e (Oxford) and processed for immunostaining usmg the p e r o x i d a s e antiperoxidase ( P A P ) complex 10. The rabbit antiserum (no. 4603-2) to NPY was used at a dilution of 1:1000. As previously r e p o r t e d , only i m m u n o a d s o r p tion with synthetic N P Y could abolish immunostaining 7. Two peptides with a high degree of homology with NPY, PYY and PP. as well as a series of hypo-
Correspondence: G. Pelletier, MRC Group in Molecular Endocrinology, Le Centre Hospitalier de l'Universit6 Laval. 2705. boul. Laurier, Ste-Foy, Quebec G1V 4G2, Que., Canada.
0006-8993/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
389 thalamic peptides including A C T H , vasoactive intestinal peptide, cholecystokinin and somatostatin were completely ineffective in blocking the reaction. In control animals, as previously described, large amounts of fibers and endings were seen in the suprachiasmatic and paraventricular nuclei as well as in the median eminence (Table I). A f t e r colchicine treatment, p e r i k a r y a were seen in large amounts in the arcuate and supraoptic nuclei. They were inconsistently found in the paraventricular nucleus. M S G t r e a t m e n t p r o d u c e d a m a r k e d decrease in the n u m b e r of immunostained cell bodies in the arcuate nucleus as evaluated after coichicine injection. The few remaining positive cell bodies were located dorsally to the arcuate nucleus. In the same animals, a striking feature was the presence of a large n u m b e r of positive cell bodies in the magnocellular portion of the paraventricular nucleus. Evaluation of the density of fibers p e r f o r m e d in animals which had been treated with M S G demonstrated a reduction of N P Y fibers in the paraventricular nucleus (Table I). As a result of M S G t r e a t m e n t , a decrease of NPY fibers, although less important, was also a p p a r e n t in the arcuate nucleus and the median eminence. No obvious change in the density of NPY fibers was observed in the other hypothalamic areas. These results strongly suggest that a large n u m b e r of N P Y fibers in the paraventricular nucleus are originating from cell bodies located in the arcuate nucleus. They are in a g r e e m e n t with recent data o b t a i n e d by Bai et al. 2 after unilateral electrolytic lesion of the arcuate nucleus. The a p p e a r a n c e of a large n u m b e r TABLE
of NPY cell bodies in the paraventricular nucleus leads to the speculation that M S G - t r e a t m e n t in the neonatal period might suppress a tonic inhibitory influence, exerted under normal conditions by arcuate neurons on the production of NPY in some paraventricular neurons. This negative feedback might be exerted by the NPY neurons of the arcuate nucleus themselves which project to the paraventricular nucleus. However, since M S G treatment induced a large non-selective destruction of arcuate neurons, it cannot be excluded that other inputs from the arcuate nucleus might be involved in this regulatory mechanism. A n other interpretation might be that the a p p e a r a n c e of NPY neurons is related to the modification of the endocrine environment resulting from the bilateral le-
I
Semiquantitative evaluation of NPY immunoreactivity in rat hypothalamus Area
Irnmunoreactivity Control
MSG-treated
Suprachiasmatic nucleus Supraoptic nucleus Paraventricular nucleus
+++++ ** * +++++
+++++ *** ++
Median eminence
0 to * + + +
*** + +
mrcuate nucleus
++
+
• ***
*
+ Fibers. * Cell bodies as evaluated in colchicine-treated animals. The intensity of staining was graded from 0 to 5.
Fig. 1. Sections through the paraventricular nucleus of colchicine-treated rats. A: rats treated with MSG. B: control rats. Cell bodies staining for NPY (arrows) are abundant in MSGtreated animals, x450.
390 sion of the arcuate nucleus 9. Such modifications might induce expression of NPY in neurons which do not produce NPY u n d e r normal conditions, suggesting neuronal genome plasticity, as it has been pro-
1 Allen, Y.S., Adrian, T.E., Allen, J.M., Tatemoto, K., Craw, T.J., Bloom, S.R. and Polack, J.M., Neuropeptide Y distribution in the rat brain, Science, 221 (1983) 877-879. 2 Bai, F.L., Yamano, M., Shiotani, Y., Emson, P.C., Smith, A.D., Powell, J.F. and Tohyama, M., An arcuato-paraventricular and -dorsomedial hypothalamic neuropeptide Ycontaining system which lacks noradrenaline in the rat, Brain Research, 331 (1985) 172-175. 3 Guy, J., Vaudry, H. and Pelletier, G., Differential projection of two immunoreactive a-melanocyte-stimulating hormone (a-MSH) neuronal systems in the rat brain, Brain Research, 220 (1981) 199-202. 4 Lemkey-Johnston, N. and Reynolds, W.E., Nature and extent of brain lesions in mice related to ingestion of monosodium glutamate, J. Neuropathol. Exp. Neurol., 231 (1974) 74-97. 5 Mezey, E. and Kiss, J.Z., Vasoactive intestinal peptidecontaining neurons in the paraventricular nucleus may participate in regulating prolactin secretion, Proc. Natl. Acad. Sci. U.S.A., 82 (1985) 245-247. 6 Obney, J.W., Brain lesions, obesity and other disturbances in mice treated with monosodium glutamate, Science, 164 (1969) 719-721. 7 Pelletier, G., D6sy, L., Kerkerian, L. and C6t6, J., Immu-
posed to explain the detection of vasopressin lI~or wlsoactive intestinal peptide 5 in the parvicellular portion of the paraventricular nucleus after adrenalectomy.
nocytochemical localization of neuropeptide Y (NPY) in the human hypothalamus, Cell Tissue Res., 238 (1984) 203-205. 8 Pelletier, G., Guy, J., Allen, Y.S. and Polak, J.M., Electron microscope immunocytochemical localization of neuropeptide Y (NPY) in the rat brain, Neuropeptides, 4 (1984) 319-324. 9 Redding, T.W., Schally, A.W., Arimura, A. and Wakayabashi, I., Effect of monosodium glutamate on some endocrine functions, Neuroendocrinology, 8 (1971) 245-255. 10 Sternberger, L.A., lmmunocytochernistry, Prentice-Hall, Englewood Cliffs, NJ, 1974, pp. 129-171. 11 Tatemoto, K., Neuropeptide Y: complete amino acid sequence of the brain peptide, Proc. Natl. Acad. Sci. U.S.A., 79 (1982) 5485-5489. 12 Tatemoto, K., Carlquist, N. and Mutt, V., Neuropeptide Y. A novel brain peptide with structural similarities to peptide YY and pancreatic polypeptide, Nature (London), 296 (1982) 654-660. 13 Wolfson, B., Manning, R.W., Davis, L.G., Arentzen, R. and Balding, F., Jr., Co-localization of corticotropin-releasing factor and vasopressin mRNA in neurones after adrenalectomy, Nature (London), 315 (1985) 59-61.
388
BRE 21~54
Effects of monosodium L-glutamate administration on neuropeptide Y-containing neurons in the rat hypothalamus L. KERKI~RIAN and G. PELLETIER MRC Group in Molecular Endocrinology, Le Centre Hospitalier de I'UniversitO Laval, Quebec G I V 4G2, Que. (Canada)
(Accepted November 26th, 1985) Key words." L-glutamate - - neuropeptide Y - - hypothalamus
Immunocytochemical localization of neuropeptide Y (NPY) was performed in the hypothalamus of rats of which the arcuate nucleus had been destroyed with monosodium L-glutamate in the neonatal period. The treatment produced a disappearance of most of the NPY cell bodies normally found in the arcuate nucleus. The concentration of fibers was decreased in the paraventricular nucleus, but not in the other hypothalamic nuclei. The treatment also induced the appearance of a large number of immunoreactive cell bodies in the paraventricular nucleus. These results strongly suggest that arcuate NPY neurons are projecting to the paraventricular nucleus and that the arcuate nucleus probably exerts some inhibitory tonic influence on NPY paraventricular neurons.
N e u r o p e p t i d e Y (NPY) is a 36-amino acid residue peptide which has been characterized from porcine brain by T a t e m o t o ~ and T a t e m o t o et al. x2. This peptide has structural analogy with the gut peptide YY (PYY) and pancreatic polypeptide (PP). Recently, Allen et al. 1 using antibodies to porcine N P Y have clearly d e m o n s t r a t e d that PP-like immunoreactivity, found in neurons of rat brain, was due to cross-reactivity of antibodies to PP with NPY. In the rat and man, NPY has been localized by immunohistochemistry in several brain areas. In the hypothalamus, numerous NPY-containing neuronal cell bodies were observed in the arcuate nucleus, whereas high concentration of immunoreaction fibers have been detected in the suprachiasmatic and paraventricular nuclei 1,7.8. A m o d e r a t e l y dense network of positive fibers was also observed in the arcuate nucleus and inner zones of the m e d i a n eminence. The origin of the NPY fibers found in nuclei of the hypothalami has not been extensively studied. Very recently, Bai et ai.2 have shown that electrical lesion in the area of the arcuate nucleus p r o d u c e d a decrease of NPY fibers ipsilaterally in the paraventricular and dorsal lateral nuclei of the rat. In o r d e r to evaluate the pro-
jection of the arcuate N P Y neurons, we p r o c e e d e d to an immunohistochemical localization of NPY after treatment with m o n o s o d i u m L-glutamate ( M S G ) which selectively destroys the arcuate nucleus 3,4.~. Seven female rats received 2.0 mg/g b. wt. on the 2nd and 4th days of life and 4.0 mg/g b. wt. on the 6th, 8th and 10th days. As control, a group of 6 female rats were similarly treated with the vehicle (saline). They were used for immunohistochemistry at 3 months of age. Three ammals p e r group were injected with colchicine (25/~g) into the left lateral ventricle 48 h before fixation. All the animals were fixed by a vascular perfusion of a solution of 4 % paraformaldehyde in 0.1 M p h o s p h a t e buffer (pH 7.4). The brains were then dissected out and i m m e r s e d in the same fixative for 18 h. Consecutive coronal sections (30/~m) were cut with the V i b r a t o m e (Oxford) and processed for immunostaining usmg the p e r o x i d a s e antiperoxidase ( P A P ) complex 10. The rabbit antiserum (no. 4603-2) to NPY was used at a dilution of 1:1000. As previously r e p o r t e d , only i m m u n o a d s o r p tion with synthetic N P Y could abolish immunostaining 7. Two peptides with a high degree of homology with NPY, PYY and PP. as well as a series of hypo-
Correspondence: G. Pelletier, MRC Group in Molecular Endocrinology, Le Centre Hospitalier de l'Universit6 Laval. 2705. boul. Laurier, Ste-Foy, Quebec G1V 4G2, Que., Canada.
0006-8993/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
389 thalamic peptides including A C T H , vasoactive intestinal peptide, cholecystokinin and somatostatin were completely ineffective in blocking the reaction. In control animals, as previously described, large amounts of fibers and endings were seen in the suprachiasmatic and paraventricular nuclei as well as in the median eminence (Table I). A f t e r colchicine treatment, p e r i k a r y a were seen in large amounts in the arcuate and supraoptic nuclei. They were inconsistently found in the paraventricular nucleus. M S G t r e a t m e n t p r o d u c e d a m a r k e d decrease in the n u m b e r of immunostained cell bodies in the arcuate nucleus as evaluated after coichicine injection. The few remaining positive cell bodies were located dorsally to the arcuate nucleus. In the same animals, a striking feature was the presence of a large n u m b e r of positive cell bodies in the magnocellular portion of the paraventricular nucleus. Evaluation of the density of fibers p e r f o r m e d in animals which had been treated with M S G demonstrated a reduction of N P Y fibers in the paraventricular nucleus (Table I). As a result of M S G t r e a t m e n t , a decrease of NPY fibers, although less important, was also a p p a r e n t in the arcuate nucleus and the median eminence. No obvious change in the density of NPY fibers was observed in the other hypothalamic areas. These results strongly suggest that a large n u m b e r of N P Y fibers in the paraventricular nucleus are originating from cell bodies located in the arcuate nucleus. They are in a g r e e m e n t with recent data o b t a i n e d by Bai et al. 2 after unilateral electrolytic lesion of the arcuate nucleus. The a p p e a r a n c e of a large n u m b e r TABLE
of NPY cell bodies in the paraventricular nucleus leads to the speculation that M S G - t r e a t m e n t in the neonatal period might suppress a tonic inhibitory influence, exerted under normal conditions by arcuate neurons on the production of NPY in some paraventricular neurons. This negative feedback might be exerted by the NPY neurons of the arcuate nucleus themselves which project to the paraventricular nucleus. However, since M S G treatment induced a large non-selective destruction of arcuate neurons, it cannot be excluded that other inputs from the arcuate nucleus might be involved in this regulatory mechanism. A n other interpretation might be that the a p p e a r a n c e of NPY neurons is related to the modification of the endocrine environment resulting from the bilateral le-
I
Semiquantitative evaluation of NPY immunoreactivity in rat hypothalamus Area
Irnmunoreactivity Control
MSG-treated
Suprachiasmatic nucleus Supraoptic nucleus Paraventricular nucleus
+++++ ** * +++++
+++++ *** ++
Median eminence
0 to * + + +
*** + +
mrcuate nucleus
++
+
• ***
*
+ Fibers. * Cell bodies as evaluated in colchicine-treated animals. The intensity of staining was graded from 0 to 5.
Fig. 1. Sections through the paraventricular nucleus of colchicine-treated rats. A: rats treated with MSG. B: control rats. Cell bodies staining for NPY (arrows) are abundant in MSGtreated animals, x450.
390 sion of the arcuate nucleus 9. Such modifications might induce expression of NPY in neurons which do not produce NPY u n d e r normal conditions, suggesting neuronal genome plasticity, as it has been pro-
1 Allen, Y.S., Adrian, T.E., Allen, J.M., Tatemoto, K., Craw, T.J., Bloom, S.R. and Polack, J.M., Neuropeptide Y distribution in the rat brain, Science, 221 (1983) 877-879. 2 Bai, F.L., Yamano, M., Shiotani, Y., Emson, P.C., Smith, A.D., Powell, J.F. and Tohyama, M., An arcuato-paraventricular and -dorsomedial hypothalamic neuropeptide Ycontaining system which lacks noradrenaline in the rat, Brain Research, 331 (1985) 172-175. 3 Guy, J., Vaudry, H. and Pelletier, G., Differential projection of two immunoreactive a-melanocyte-stimulating hormone (a-MSH) neuronal systems in the rat brain, Brain Research, 220 (1981) 199-202. 4 Lemkey-Johnston, N. and Reynolds, W.E., Nature and extent of brain lesions in mice related to ingestion of monosodium glutamate, J. Neuropathol. Exp. Neurol., 231 (1974) 74-97. 5 Mezey, E. and Kiss, J.Z., Vasoactive intestinal peptidecontaining neurons in the paraventricular nucleus may participate in regulating prolactin secretion, Proc. Natl. Acad. Sci. U.S.A., 82 (1985) 245-247. 6 Obney, J.W., Brain lesions, obesity and other disturbances in mice treated with monosodium glutamate, Science, 164 (1969) 719-721. 7 Pelletier, G., D6sy, L., Kerkerian, L. and C6t6, J., Immu-
posed to explain the detection of vasopressin lI~or wlsoactive intestinal peptide 5 in the parvicellular portion of the paraventricular nucleus after adrenalectomy.
nocytochemical localization of neuropeptide Y (NPY) in the human hypothalamus, Cell Tissue Res., 238 (1984) 203-205. 8 Pelletier, G., Guy, J., Allen, Y.S. and Polak, J.M., Electron microscope immunocytochemical localization of neuropeptide Y (NPY) in the rat brain, Neuropeptides, 4 (1984) 319-324. 9 Redding, T.W., Schally, A.W., Arimura, A. and Wakayabashi, I., Effect of monosodium glutamate on some endocrine functions, Neuroendocrinology, 8 (1971) 245-255. 10 Sternberger, L.A., lmmunocytochernistry, Prentice-Hall, Englewood Cliffs, NJ, 1974, pp. 129-171. 11 Tatemoto, K., Neuropeptide Y: complete amino acid sequence of the brain peptide, Proc. Natl. Acad. Sci. U.S.A., 79 (1982) 5485-5489. 12 Tatemoto, K., Carlquist, N. and Mutt, V., Neuropeptide Y. A novel brain peptide with structural similarities to peptide YY and pancreatic polypeptide, Nature (London), 296 (1982) 654-660. 13 Wolfson, B., Manning, R.W., Davis, L.G., Arentzen, R. and Balding, F., Jr., Co-localization of corticotropin-releasing factor and vasopressin mRNA in neurones after adrenalectomy, Nature (London), 315 (1985) 59-61.