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Enkephalin-like immunoreactive neurons and fibers in the ventromedial hypothalamic nucleus
Brain Research Bulletin, Vol. 17, pp. 15%167, 1986. o Ankho International
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Enkeph~in-Lee Immunoreactive Neurons and Fibers in the Ventromedial Hypothalamic Nucleus S. INAGAKI,* Y. KUBOTA,* S. KITO,” M. YAMANOt AND M. TOHYAMAt *Third Department TDepartment
of Internal Medicine, Hiroshima University School of Medicine, l-2-3 Kasumi Minami-ku, Hiroshima 734 Japan of Neuroanatomy, Institute of Higher Nervous Activity, Osaka University Medical School Nakanoshima, Kitaku, Osaka 530 Japan
Received 25 March 1986 INAGAKI,
S., Y. KUBOTA, S. KITO, M. YAMANO AND M. TOHYAMA. Enkepahlin-like immunoreuctive neurons hypothulamic nucleus. BRAIN RES BULL 17(2) 159-167, 1986.-Enkephalin-like im-
undfibers in the wntromediul
munoreacitve (ENK-IR) neurons and fibers in the rat ventromedial hypothalamic nucleus (VMH) were examined by light and electron microscopy using the peroxidase-antiperoxidase immunocytochemistry. There were groups of ENK-IR neurons present in the ventrolateral part of the VMH, and such neurons were scattered elsewhere. These neurons had perikarya 10-Z pm in diameter with moderately developed cell organelles and enfolded nuclei that were often distributed eccentrically placed in the cell. The perikarya and dendrites contained diffuse, large-cored vesicles (LCV) (6&230 nm with a p~~rnin~ce in the 60-80 nm). ENK-IR neurons received synaptic inputs on the soma and dendrites from u&&&d axonal boutons containing many small, clear vesicles and occasional LCV. The u&structural features of the ENK-IR cells in the VMH seemed to correspond to the “common cells” described by Millhouse. Dense ENK-IR fibers were distributed in this nucleus throughout the rostrocaudal and ventrodorsal areas. Axonal boutons containing numerous small, clear vesicles, and dispersed LCV generally made synapticcontacts with the cell bodies and dendritesof unlabeledneurons. The findingssuggestthat opioid peptides directly influence VMH neurons through synaptic contacts. Enkephalin
Immunohistochemistry
Ventromedial hypothalamic nucleus
OPIOID peptides such as morphine and the enkephalins increase the neuronal activity of the ventromedial hypothalamic nucleus (VMH) [2,9], including that of glucoreceptor neurons [ 181. In fact, enkephalin-like immunoreactive (ENK-IR) neurons and fibers are present in large numbers in the VMH [3, 4, 23, 241, suggesting that endogenous enkephalin (ENK) affects VMH neurons through synaptic contacts. This nucleus is the satiety center; there, lesions cause hy~~~~a 111,electric stimulation suppresses food intake [10,161, and chemical stimulation induces glucogenolysis in the liver [IS]. Thus, ENK-containing neurons and fibers in the VMH might be involved in its control of feeding. However, little is known as to the tine structure and synaptic organization of ENK-IR structures in the VMH. In view of the important role that enkephalin may have in the VMH, we investigated by the peroxidaseantiperoxidase (PAP) technique [Zl] the lit microscopic and ult~s~ctural localizations including synaptic organization of ENK-IR structures in the VMH.
Rat
Electron microscopy
untreated animals were used for observation of ENK-IR fibers and another five were treated with a lateral-ventricular administration of colchicine (50 &O ~1) to enhance ENK immunoreactivity in the perikarya. All animals, treated and untreated, were perfused intracardiaIly with saline and then with the fixation of Somogyi and Takagi [20]. Brains were removed and immersed in the same fixative for 2 hr at 4°C. After being washed in 0.1 M phosphate buffer (PH 7.4), frontal sections 60 pm thick were cut on a Vibratome and left overnight at 4°C in 0.1 M phosphate buffer (pH 7.4). Immunohistochemistry The immunohistochemical procedures were described elsewhere [6]. Sections of the VMH were incubated in 10% normal goat serum in phosphate-buffered saline (PBS) for 60 min at 2WC, then incubated overnight at 4°C in Met- or L.eu-ENK ~ti~~rn diluted 1:2000. The buffer for dilution and washing was PBS containing 1% normal goat serum. As the control, Met- or Leu-ENK antiserum was absorbed with synthetic Met- or Leu-ENK at 1x lo-“’ M. The sections were washed three times in PBS, incubated with goat anti-rabbit IgG diluted 1:100 overnight at 2o”C, and then incubated with rabbit PAP complex (DAKO) diluted 1:200 for 2 hr at 20°C. The tissue was washed twice in PBS and twice in Tris-HCl
METHOD
Animals and Tissue Preparation
Adult male Wistar rats weighing 120-200 g were used. Six
159
160
FIG. 1. Light micrograph ofenkephalin-like immunareactive (ENK-IR) structures in the ventral half of the ventromedial hypothalamic nucleus (VMH) of a colchicine treated rat. Groutx of ENK-IR neurons are seen in the ventrolateral portion of the VMH. Scale: 50 brn.
FIG. 2. A schematical drawing showing the distribution of ENR-IR neurons in the VMH of the middle level on the frontal plane. III, third ventricle.
ELECTRON MICROSCOPY OF VMH ENK NEURONS
161
FIG. 3. (A) Electron micrographsof ENK-IR cell bodies which are in synaptic contact with unlahekd axon terminals. These neurons have k+qe enfolded nuclei and immunostained cytoplasmk [email protected] cytoplasm contained mitochondria, a few shott segments of rER, and Golgi conrpk~es (G). Large cored vesicles (LCV), which are densely iareseeainthep&pberyofthepetikarya (small arrows). (B) is a hi& power view of axe-somatic contacts (thick at~~ws) of (A) h&&en the ENK-IR c&s and unhbekd axonal houtons. Unlabekd axonal boutons contain numerous small clear vesicles and mitochondria. In (B), a LCV appearsto be asso&ed with a Golgi complex (0) (lowest arrow). Small arrows show LCV either with or without immunoprecipitation.Scales: (A) 1 pm; B, 0.5 pm.
buffer @H 7.6); it was reacted for 5-15 min in 0.2 m&ml
3,3’-diaminobenxidine-4HCl (DOTITE) dissolved in 50 mM Tris-HCl buf&r containing 0.005% hydrogen peroxidase, washed, and postfixed for 1 hr in 1% 0~0, in 0.1 M phosphate buffer (pH 7.4). The sections were dehydrated, stained with 1% uranyl acetate in a 70% alcohol dehydrated state,
and flat-embedded on slides in Epon 812. Electron micrographs were taken at 75 kV on a Hitachi H-600 electron microscope. Antisera
The preparation and characteristics of the rabbit antisera
FIG. 4. (A) Electron micrographs of ENK-IR cell bodies which are in synaptic contact with unlabeled axon terminals. I’hcse neu~-on\ have large enfolded nuclei and immunostained cytoplasmic matrix. The cytoplasm contained mitochondria. a few short bcgmcnts ot rER, and Golgi complexes (G). Large cored vesicles (LCV), which are densely immunostained, are seen in the periphery of the perikarya (small arrows). A chromatoid body (c) is seen in ENK-IR perikarya. (B) is high power view of axo-somatic contact> (thick arrows) of (A) between the ENK-IR cells and unlabeled axonal boutons. Unlabeled axonal boutons contain numerous small clear vesicles and mitochondria. (C) shows another ENK-IR neuron receiving synaptic input (*) from an unlabeled axon terminal which contain numerous small clear vesicles and two LCV (small arrows). Scales: (A) I pm: (B) and (C) 0.1 /em.
ELECTRON
MICROSCOPY
163
OF VMH ENK NEURONS
FIG. 5. Electron micrographs of ENK-IR dendrites receiving synaptic inputs from unlabeled axonal boutons. Immunostained dendrites, some of which contain scattered LCV (arrow) (A), frequently make asymmetrical synaptic contacts with unlabeled axon terminals containing numerous small clear vesicles (A,B) and sparse LCV (arrow) (B). Asterisks show immunoreactive dendrites. Scales: 0.1 pm.
to Met- and Leu-ENK were described elsewhere [5,23]. In brief, the antisera were produced in rabbits in response to immunogens made by linking either synthetic Met-ENK or Leu-ENK (Peptide Laboratory) to bovine thyroglobulin (Sigma). The Met-ENK antiserum cross-reacts 4% with Leu-ENK and 0.56% with Met-ENK-Args, but does not significantly react with Met-ENK-A&-Phe’, Leu-ENK-Arg6, dynorphin (l-13), or (l-17), or P-endorphin. The Leu-ENK antiserum cross-reacts 0.25% with Met-ENK, but does not react significantly with Leu-ENK-A@, Met-ENK-Args, dynorphin (l-13), dynorphin (l-17), or /3-endorphin. Incubation with the antiserum absorbed with synthetic Met- or Leu-ENK completely abolished the specific staining at a concentration of 1 x 10v6 M. No significant differences were seen between structures immunostained with MetENK and Leu-ENK antisera in the VMH. Since slight cross-reaction with Leu-ENK or Met-ENK was noted in immunohistochemical tests, the immunostaining observed here will be referred to as ENK-like immunoreactivity.
RESULTS ENK-IR
Neurons
Light microscopy. Groups of ENK-IR cells that were small to medium in size (10-25 pm along the long axis) were seen in the ventrolateral portion of the VMH of colchicine treated rats (Figs. 1, 2). Some of these neurons were continuous with ENK-IR neurons just ventrolateral to the VMH. Neurons were scattered elsewhere, particularly in the periphery of the VMH (Fig. 2). Immunoreactive neurons were of various shapes, such as round, ovoid, fusiform, and multipolar. They usually had one or two principal dendrites; less often they had three, and rarely four. The exact number of dendrites of ENK-IR neurons may be greater than we
found, since immunostaining depends on the location and quantity of immunogens and on the titers of the antisera. Some dendrites may have been cut during preparation of the sections 60 pm thick. Electron microscopy. VMH neurons stained with ENK antisera in the ventrolateral part and elsewhere had similar cytological features. We mainly examined ENK-IR neurons in the ventrolateral part ae the electron microscopic level, since they were numerous there. The neurons had perikarya with moderately developed cell organelles (Figs. 3A, 4A). Most ribosomes were unbound rosettes, and unevenly distributed in the cytoplasm. Rough endoplasmic reticulum (rER) was most often seen as single, short strands. Compared to the rER, the Golgi apparatus was more prominent (Figs. 3, 4A). Although this organelle was seen almost everywhere in the cell body, it was particularly conspicuous in the perinuclear area. Large-cored vesicles (LCV; 60-230 nm) were found in the cell bodies and dendrites with a predominance of LCV in the 60-80 nm (Figs. 3,4A, 5A). In the perikarya, LCV were more frequently seen in the periphery, which were sometimes associated with Golgi apparatus (Figs. 3, 4A). Nuclei had enfoldings and in many cases were eccentric. In parts of the cytoplasm opposite to eccentrically placed nuclei, abundant cell organelles such as mitochondria, rER, free ribosomes, and Golgi apparatus were sometimes seen. Chromatoid bodies like those described by Millhouse 1171 were occasionally seen in ENK-IR perikarya (Fig. 4A). The peroxidase reaction product was scattered throughout the cytoplasm and heavily precipitated over the cores of LCV (Figs. 3, 4A). The proximal (l-2 pm in diameter) and distal dendrites (0.5-l pm in diameter) of labeled neurons also contained occasional LCV (60-100 nm) heavily labeled with peroxidase reaction product (Fig. 5A). ENK-IR neurons in the VMH received synaptic inputs
FIG. 6. Electron micrographs of ENK-IR axon terminals which are in synaptic contact with unlabeled dendrites in the VMH, lmmunoprecipitate is seen prominently in the LCV (A,C,D). and through the axoplasma. Smail clear vesicles are packed in ENK-IR terminals, particularly prominent in a bouton shown in (Bf. These axons frequently make synaptic contacts with unlabeled dendritic shafts (A,B) and dendritic spines (C,D). A typical citemal oganelle is seen at the neck of a spine (C). Most of these contacts are asymmetrical (A.C,D). Double smalf arrows show postjunctiooai dense body (D). Scales: 0. I pm.
from unlabeled boutons on their somata (Figs. 3, 4) and dendrites (Fig. 5). These unlabeled boutons contained many small, clear vesicles 30-50 nm in diameter (Figs, 3B, 4B,4C, 5), which were round, oval, or flattened with or without occasional LCV. The unlabeied axonal boutons very frequently made synaptic contacts with ENK-IR dendrites (Fig. 5), and less frequently with soma of ENK-IR neurons (Figs. 3B, 4B, 4C). Axo-dendritic synapses were most frequentIy asymmetrical, while axo-somatic synapses were primarily symmetrical.
IiNK-IA
Axons
Light ~is~~s~~~~~. A dense plexus of ENK-IR fibers were distributed in the VMH of untreated animds throughout the entire rostrocaudal and dorsoventral areas. The location, seen by light microscope, of ENK-IR fibers in the VMH was described in detail elsewhere [4, 23, 243. Elrctrtm microscopy. ENK-IR fibers were generally unmyelinated, axons (0.1-0.5 wrn in diameter) and axonal boutons (0.4-1.2 pm in diameter) were seen throughout the
165
ELECTRON MICROSCOPY OF VMH ENK NEURGNS
FIG. 7. Electron micrographs of an unlabeled VMN neuron which is in symmetrical synaptic contact with BNK-IR axonal boutons (thick and thin arrows). (B) is a high power view of a part of(A) (thick arrows). Asterisksshow ENK-IRaxonalboutons (B). Scales:(A) 1pm; (B) 0.1 pm.
VMH. These axonal boutons contained many small, clear vesicles (30-50 nm) and occasional LCV in which a heavy precipitate of peroxidase reaction product was visible (Fig. 6). Immunopr~ipi~te was diffused th~~~out the axeplasma, and was seen on the membranes of cell organelles. ENK-IR +xonal boutons formed synapses with dendritic shafts (Fig. 6 A and B), dendritic spines (Fig. 6 C and D), aad less frequently with the soma of unlabeled ne~ons (Fig. 7). Most of the neurons postsynaptic to ENK-IR axons were IO-25 Frn along the long axis and had a relatively sparse ~p~tion of LCV (60-230 nm with a predominance in the 6os(o run). No mtied differences were found between these neurons and ENK-IR neurons in the VMH. Axo-somatic synapses were mostly symmetrical (Fig. 7B) and axodendritic synapses were most frequently asymmetrical (Fig. 6 A, C, D). Labeled boutons were occasionally in apposition to the labeled neuronal soma and dendrites but rarely made axe-somatic synapses (data not shown).
DISCUSSION The present fmdings showed that the rat VMH contained many ENK-IR neurons. Dense plexuses of ~muno~ve fibers in the VMH formed synapses with various neuronal elements. The Golgi method has shown two kinds of neurons in the VMH [17]. Most are Type I cells with soma 10-30 pm across and 2-3 principal dendrites. The Type II cells seem to make up no more than 14% of the neuronal population; they were mostly in the lateral part of the VMH, and had larger soma 30-50 w in diameter. bud 1171has also divided VMH neurons into two classes on ultrastructural observacells.” Most are the tions, “common” and “aeon “common cells” that contain short segments of rER, rosettes of unbound ribosomes, and chromatoid bodies. They frequently receive synaptic inputs on the soma. The “anco~on cells” are larger with more knot rER and Nissl bodies. The “common cds” seem to correspond with
Type I cells and the “uncommon cells” with Type II [17]. Our findings have suggested that ENK-IR neurons were the “common cells,” since ENK-IR neurons are 10-25 pm across, and have single, short strands of rER, rosettes of unbound ribosomes, a few small Nissl bodies, and frequent synaptic contacts on the soma. Light microscopic observation of ENK-IR cells suggested that ENK-IR neurons and Type II cells were more or less in the same place, in the ventrolateral portion of the VMH. However, ultrastructural features of ENK-IR neurons corresponded to the “common cells,” which seem to be Type I cells by Golgi impregnation. Yamano rr ul. [23] found, using immunohistochemistry combined with knife cuts, electric lesion, and retrograde tracers, that enkephalinergic fibers in the VMH largely originate from the paraventricular hypothalamic nucleus. ENK-IR fibers in the VMH markedly decrease in most areas of the VMH after a lesion is made in the paraventricular nucleus, while most fibers in the ventrolateral part remain intact [23]. Thus, most ENK-IR fibers in the VMH except the ventrolateral area, arise from paraventricular ENK-IR neurons, while most fibers in the ventrolateral part of the VMH probably arise from locally synapsing axons belonging to ENK-IR neurons in the VMH. These fibers formed synaptic contacts with the dendrites and soma of VMH neurons. Perhaps the effect of opioids on the neuronal activity of VMH neurons [2,9], including glucoreceptor VMH neurons [18], is regulated by synapsing axons originating from ENK-IR paraventricular neurons or intrinsic VMH neurons. Ono er ~1. [18l showed that the direct excitatory &&%f
[ 121. Both of these manipulations interfere with enkephalin, which is perhaps serving as an excitatory transmitter or neuromodulator, mimics the excitatory effect of glucose in the VMH [ 191, so our results are in exact agreement with theirs. ENK-IR neurons scattered in the dorsal part of the VMH give rise to axons that innervate the midbrain central gray matter [25]. The midbrain central gray matter, where dense ENK-IR fibers are seen, is known to be a component of an opioid-mediated pain-suppression system 1131. Thus, ENK-IR neurons projecting to the midbrain central gray matter may modulate the neuronal activity in this area. in association with an analgesic system. While the VMH rcceives major inputs from the amygdala, the parabrachial nucleus [14], and the rostra1 hypothalamus. including the preoptic area [22]. Various neuropeptides are contained in these afferent systems. For example, neurotensin is found in neuronal pathways from the amygdala to the VMH 14.71. cholecystokinin in parabrachial-VMH pathways (4, 6, 261, and ENK in paraventriculo-VMH pathways 14.231. Thus, these neuropeptides in the VMH may be involved in regulation of various VMH functions such as feeding [I. I& 121, motivation [16], analgesia [13,25], and reproduction [8] in relation to the neuronal activity in the various brain areas.
ACKNOWLEDGEMENTS
The authors wish to thank Dr. Y. Shiotani (Department of Neuroanatomy, Institute of Higher Nervous Activity, Osaka University Medical School, Osaka, Japan) and Dr. T. Ono (Department of Physiology, Faculty of Medicine, Toyama Medical and Pharmaceutical University) for the valuable and critical comments. This work was supported by Grant-in-Aid for Scientific Research from the Japanese Ministry of Education.
REFERENCES 1. Anand, B. K. and J. R. Brobeck. Hypothalamic control of food intake in rats and cats. Yale J Biol Mrd 24: 123-140, 1951.
2. Eidelberg, E. and M. L. Bond. Effects of morphine and antagonists on hypothalamic activity. Arch Iut Pharmacx~dyrr 196: 16-24, 1972. 3. Elde, R. P., T. Hokfelt, 0. Johansson and L. Terenius. lmmunohistochemical studies using antibodies to leucineenkephalin: initial observations on the nervous system of the rat. Neuroscience 1: 349-351, 1976. 4. Inagaki, S., S. Kito, Y. Kubota, M. Yamano, M. Tohyama and Y. Shiotani. Experimental and immunohistochemcial studies on neuropeptides in the nucleus ventromedialis hypothalami of the rat. In: Gunma Symposia Endocrinology. vol 22, edited by M. Suzuki. Tokyo: Center Academic Publication JPN, 1985. pp. 171-182. of enkephalin5. Inagaki, S. and A. Parent. Distribution immunoreactive neurons in the forebrain and upper brainstem of the squirrel monkey. Brain Res 359: 267-280; i985. 6. lnaeaki. S.. Y. Shiotani. M. Yamano. S. Shiosaka. H. Takaai. K. ?ateishi; E. Hashimura, T. Hamaoka and M. Tohyama. Distribution, origin, and fine structures of cholecystokinin-8-like immunoreactive terminals in the nucleus ventromedialis hypothalami of the rat. J Nrumsci 4: 1289-1299, 1984. 7. lnagaki, S., M. Yamano, S. Shiosaka, H. Takagi and M. Tohyama. Distribution and origins of neurotensin containing fibers in the nucleus ventromedtahs hypothalaml of the rat: an experimental immunohistochemical study. Brain Res 273: 229-235, 1983.
8. Kennedy, G. C. Hypothalamic control of energy balance and reproductive cycle in rat. J Physiol 166: 395-407, 1963. 9. Kerr, F. W. L., J. N. Triplett, Jr. and G. W. Beeler. Reciprocal (push-pull) effects of morphine on single units in the ventromedian and lateral hypothalamus and influences in other nuclei: with a comment on methadone effects during withdrawal from morphine. Brain Res 74: 81-103, 1974. 10. Larsson, S. On the hypothalamic organization of the nervous mechanism regulating food intake. Ac,fa Physiol Stand 32: Suppl 115, l-63, 1954. 11. Leibowitz, S. F., N. J. Hammer and K. Chang. Hypothalamic paraventricular nucleus lesions produce overeating and obesity in the rat. Physic>/ Behat’ 27: 1031-1040, 1981. nucleus: A primary site 12. Leibowitz, S. F. Paraventricular mediating adrenergic stimulation of feeding and drinking. Pharmacol B&hem Behat] 8: 163-175, 1978. 13. Lewis, V. A. and C. F. Gebhart. Evaluation of the periaqueductal central grey (PAG) as a morphine specific locus of action and examination of morphine-induced and stimulation-produced analgesia at coincident PAG loci. Brain Res 124: 283-303, 1977. 14. McBride, R. L. and S. Jerome. Amygdaloid and pontine Projections to the ventromedial nucleus of the hypothalamus. .I Comp Neural
174: 377-396,
1977.
15. Matsushita, H. and T. Shimazu. Chemical coding of the hypothalamic neurons in metabolic control. Il. Norepinephrinesensitive neurons and glycogen breakdown in liver. Brain Rc.) 183: 79-87,
1980.
167 16. Miller, N. E. Motivationa\ effects of brain stimulation and drugs. Fed Proc 19: 846854, 1960. 17. MilIhouse, 0. E. Cytological observations on ventromedial hypothalamic nucleus. Ceff Tissue Res 191: 47U91, 1978. 18. Ono, T., Y. Gomura, H. Nishino, K. Sasaki, K. Muramoto and I. Yano. Morphine and enkephahn effects on hypothalamic glueoresponaive neurons. Brain Res 185: 208-212, 1980. 19. Oomura, Y., M. Ohta, S. Ishibashi, H. Kita, T. Okqjima and T. Ono. Activity of Chemosensftfve Neurons Related to Neurophysiofogical Mechanism ofFeeding, Vof 2, Recent Advances in Obesity Research, edited by G. Bray. London: Newman
Publishing, 1978, pp. 17-28. 20. Somogyi, P. and H. Takagi. A note on the use of picric acid~o~~ehyde fixative for correlated light and electron microscopic i~uno~ytochemist~. Neuroscience 7: 1779-1784, 1982. 21. Sternberger, L. A. ~mmunobfstochemfst~. Enghwood Cliffs, NJ: Prentice Hall, 1974. 22. Swanson, L. W. An auto~di~~ph~c study of the efferent connections of the preoptic region of the rat. J Comp Neurol 167: 227-256, 1976.
23. Yamano, M., S. Ina@i, S. Kito and hf. Tohyama. An enkcphalinergic projection from the hypothalamic paraventricular nucleus to the hypothalamic venhwmxfiat nucleus of the rat. An experimental imm~ohist~hemic~ study. Brain Res 331: 25-33, 1985. 24. Yamano, M., S. Inatzaki. K. Tateishi. T. Hamaoka and M. Tohyama. Ontogeny of ~europeptides in the nucleus ventromedialis had of the rat: an i~u~hist~he~c~ analysis. Dev Brain Res 16: 253-262, 1984. 25. Yamano, M., S. Inagaki, S. Kito, T. Matsuzaki, Y. Shinohara and M. Tohyama. Enkephalinergic projection from the ventromedial hypothalamic nucleus to the midbrain central gray matter in the rat: an immunocytochemical analysis. Exp Brain Res, in press. 26. Zaborszky, L., hi. C. Beinfeld, M. Palkovits and L. Heimer. Brainstem projection to the hypothalamic ventromedial nucleus in the rat: a CCK-containing long ascending pathway. Brain Res 303: 225-231, 1984.
0361~9230/86 $3.00 -t .W
Inc. Printed in the U.S.A.
Enkeph~in-Lee Immunoreactive Neurons and Fibers in the Ventromedial Hypothalamic Nucleus S. INAGAKI,* Y. KUBOTA,* S. KITO,” M. YAMANOt AND M. TOHYAMAt *Third Department TDepartment
of Internal Medicine, Hiroshima University School of Medicine, l-2-3 Kasumi Minami-ku, Hiroshima 734 Japan of Neuroanatomy, Institute of Higher Nervous Activity, Osaka University Medical School Nakanoshima, Kitaku, Osaka 530 Japan
Received 25 March 1986 INAGAKI,
S., Y. KUBOTA, S. KITO, M. YAMANO AND M. TOHYAMA. Enkepahlin-like immunoreuctive neurons hypothulamic nucleus. BRAIN RES BULL 17(2) 159-167, 1986.-Enkephalin-like im-
undfibers in the wntromediul
munoreacitve (ENK-IR) neurons and fibers in the rat ventromedial hypothalamic nucleus (VMH) were examined by light and electron microscopy using the peroxidase-antiperoxidase immunocytochemistry. There were groups of ENK-IR neurons present in the ventrolateral part of the VMH, and such neurons were scattered elsewhere. These neurons had perikarya 10-Z pm in diameter with moderately developed cell organelles and enfolded nuclei that were often distributed eccentrically placed in the cell. The perikarya and dendrites contained diffuse, large-cored vesicles (LCV) (6&230 nm with a p~~rnin~ce in the 60-80 nm). ENK-IR neurons received synaptic inputs on the soma and dendrites from u&&&d axonal boutons containing many small, clear vesicles and occasional LCV. The u&structural features of the ENK-IR cells in the VMH seemed to correspond to the “common cells” described by Millhouse. Dense ENK-IR fibers were distributed in this nucleus throughout the rostrocaudal and ventrodorsal areas. Axonal boutons containing numerous small, clear vesicles, and dispersed LCV generally made synapticcontacts with the cell bodies and dendritesof unlabeledneurons. The findingssuggestthat opioid peptides directly influence VMH neurons through synaptic contacts. Enkephalin
Immunohistochemistry
Ventromedial hypothalamic nucleus
OPIOID peptides such as morphine and the enkephalins increase the neuronal activity of the ventromedial hypothalamic nucleus (VMH) [2,9], including that of glucoreceptor neurons [ 181. In fact, enkephalin-like immunoreactive (ENK-IR) neurons and fibers are present in large numbers in the VMH [3, 4, 23, 241, suggesting that endogenous enkephalin (ENK) affects VMH neurons through synaptic contacts. This nucleus is the satiety center; there, lesions cause hy~~~~a 111,electric stimulation suppresses food intake [10,161, and chemical stimulation induces glucogenolysis in the liver [IS]. Thus, ENK-containing neurons and fibers in the VMH might be involved in its control of feeding. However, little is known as to the tine structure and synaptic organization of ENK-IR structures in the VMH. In view of the important role that enkephalin may have in the VMH, we investigated by the peroxidaseantiperoxidase (PAP) technique [Zl] the lit microscopic and ult~s~ctural localizations including synaptic organization of ENK-IR structures in the VMH.
Rat
Electron microscopy
untreated animals were used for observation of ENK-IR fibers and another five were treated with a lateral-ventricular administration of colchicine (50 &O ~1) to enhance ENK immunoreactivity in the perikarya. All animals, treated and untreated, were perfused intracardiaIly with saline and then with the fixation of Somogyi and Takagi [20]. Brains were removed and immersed in the same fixative for 2 hr at 4°C. After being washed in 0.1 M phosphate buffer (PH 7.4), frontal sections 60 pm thick were cut on a Vibratome and left overnight at 4°C in 0.1 M phosphate buffer (pH 7.4). Immunohistochemistry The immunohistochemical procedures were described elsewhere [6]. Sections of the VMH were incubated in 10% normal goat serum in phosphate-buffered saline (PBS) for 60 min at 2WC, then incubated overnight at 4°C in Met- or L.eu-ENK ~ti~~rn diluted 1:2000. The buffer for dilution and washing was PBS containing 1% normal goat serum. As the control, Met- or Leu-ENK antiserum was absorbed with synthetic Met- or Leu-ENK at 1x lo-“’ M. The sections were washed three times in PBS, incubated with goat anti-rabbit IgG diluted 1:100 overnight at 2o”C, and then incubated with rabbit PAP complex (DAKO) diluted 1:200 for 2 hr at 20°C. The tissue was washed twice in PBS and twice in Tris-HCl
METHOD
Animals and Tissue Preparation
Adult male Wistar rats weighing 120-200 g were used. Six
159
160
FIG. 1. Light micrograph ofenkephalin-like immunareactive (ENK-IR) structures in the ventral half of the ventromedial hypothalamic nucleus (VMH) of a colchicine treated rat. Groutx of ENK-IR neurons are seen in the ventrolateral portion of the VMH. Scale: 50 brn.
FIG. 2. A schematical drawing showing the distribution of ENR-IR neurons in the VMH of the middle level on the frontal plane. III, third ventricle.
ELECTRON MICROSCOPY OF VMH ENK NEURONS
161
FIG. 3. (A) Electron micrographsof ENK-IR cell bodies which are in synaptic contact with unlahekd axon terminals. These neurons have k+qe enfolded nuclei and immunostained cytoplasmk [email protected] cytoplasm contained mitochondria, a few shott segments of rER, and Golgi conrpk~es (G). Large cored vesicles (LCV), which are densely iareseeainthep&pberyofthepetikarya (small arrows). (B) is a hi& power view of axe-somatic contacts (thick at~~ws) of (A) h&&en the ENK-IR c&s and unhbekd axonal houtons. Unlabekd axonal boutons contain numerous small clear vesicles and mitochondria. In (B), a LCV appearsto be asso&ed with a Golgi complex (0) (lowest arrow). Small arrows show LCV either with or without immunoprecipitation.Scales: (A) 1 pm; B, 0.5 pm.
buffer @H 7.6); it was reacted for 5-15 min in 0.2 m&ml
3,3’-diaminobenxidine-4HCl (DOTITE) dissolved in 50 mM Tris-HCl buf&r containing 0.005% hydrogen peroxidase, washed, and postfixed for 1 hr in 1% 0~0, in 0.1 M phosphate buffer (pH 7.4). The sections were dehydrated, stained with 1% uranyl acetate in a 70% alcohol dehydrated state,
and flat-embedded on slides in Epon 812. Electron micrographs were taken at 75 kV on a Hitachi H-600 electron microscope. Antisera
The preparation and characteristics of the rabbit antisera
FIG. 4. (A) Electron micrographs of ENK-IR cell bodies which are in synaptic contact with unlabeled axon terminals. I’hcse neu~-on\ have large enfolded nuclei and immunostained cytoplasmic matrix. The cytoplasm contained mitochondria. a few short bcgmcnts ot rER, and Golgi complexes (G). Large cored vesicles (LCV), which are densely immunostained, are seen in the periphery of the perikarya (small arrows). A chromatoid body (c) is seen in ENK-IR perikarya. (B) is high power view of axo-somatic contact> (thick arrows) of (A) between the ENK-IR cells and unlabeled axonal boutons. Unlabeled axonal boutons contain numerous small clear vesicles and mitochondria. (C) shows another ENK-IR neuron receiving synaptic input (*) from an unlabeled axon terminal which contain numerous small clear vesicles and two LCV (small arrows). Scales: (A) I pm: (B) and (C) 0.1 /em.
ELECTRON
MICROSCOPY
163
OF VMH ENK NEURONS
FIG. 5. Electron micrographs of ENK-IR dendrites receiving synaptic inputs from unlabeled axonal boutons. Immunostained dendrites, some of which contain scattered LCV (arrow) (A), frequently make asymmetrical synaptic contacts with unlabeled axon terminals containing numerous small clear vesicles (A,B) and sparse LCV (arrow) (B). Asterisks show immunoreactive dendrites. Scales: 0.1 pm.
to Met- and Leu-ENK were described elsewhere [5,23]. In brief, the antisera were produced in rabbits in response to immunogens made by linking either synthetic Met-ENK or Leu-ENK (Peptide Laboratory) to bovine thyroglobulin (Sigma). The Met-ENK antiserum cross-reacts 4% with Leu-ENK and 0.56% with Met-ENK-Args, but does not significantly react with Met-ENK-A&-Phe’, Leu-ENK-Arg6, dynorphin (l-13), or (l-17), or P-endorphin. The Leu-ENK antiserum cross-reacts 0.25% with Met-ENK, but does not react significantly with Leu-ENK-A@, Met-ENK-Args, dynorphin (l-13), dynorphin (l-17), or /3-endorphin. Incubation with the antiserum absorbed with synthetic Met- or Leu-ENK completely abolished the specific staining at a concentration of 1 x 10v6 M. No significant differences were seen between structures immunostained with MetENK and Leu-ENK antisera in the VMH. Since slight cross-reaction with Leu-ENK or Met-ENK was noted in immunohistochemical tests, the immunostaining observed here will be referred to as ENK-like immunoreactivity.
RESULTS ENK-IR
Neurons
Light microscopy. Groups of ENK-IR cells that were small to medium in size (10-25 pm along the long axis) were seen in the ventrolateral portion of the VMH of colchicine treated rats (Figs. 1, 2). Some of these neurons were continuous with ENK-IR neurons just ventrolateral to the VMH. Neurons were scattered elsewhere, particularly in the periphery of the VMH (Fig. 2). Immunoreactive neurons were of various shapes, such as round, ovoid, fusiform, and multipolar. They usually had one or two principal dendrites; less often they had three, and rarely four. The exact number of dendrites of ENK-IR neurons may be greater than we
found, since immunostaining depends on the location and quantity of immunogens and on the titers of the antisera. Some dendrites may have been cut during preparation of the sections 60 pm thick. Electron microscopy. VMH neurons stained with ENK antisera in the ventrolateral part and elsewhere had similar cytological features. We mainly examined ENK-IR neurons in the ventrolateral part ae the electron microscopic level, since they were numerous there. The neurons had perikarya with moderately developed cell organelles (Figs. 3A, 4A). Most ribosomes were unbound rosettes, and unevenly distributed in the cytoplasm. Rough endoplasmic reticulum (rER) was most often seen as single, short strands. Compared to the rER, the Golgi apparatus was more prominent (Figs. 3, 4A). Although this organelle was seen almost everywhere in the cell body, it was particularly conspicuous in the perinuclear area. Large-cored vesicles (LCV; 60-230 nm) were found in the cell bodies and dendrites with a predominance of LCV in the 60-80 nm (Figs. 3,4A, 5A). In the perikarya, LCV were more frequently seen in the periphery, which were sometimes associated with Golgi apparatus (Figs. 3, 4A). Nuclei had enfoldings and in many cases were eccentric. In parts of the cytoplasm opposite to eccentrically placed nuclei, abundant cell organelles such as mitochondria, rER, free ribosomes, and Golgi apparatus were sometimes seen. Chromatoid bodies like those described by Millhouse 1171 were occasionally seen in ENK-IR perikarya (Fig. 4A). The peroxidase reaction product was scattered throughout the cytoplasm and heavily precipitated over the cores of LCV (Figs. 3, 4A). The proximal (l-2 pm in diameter) and distal dendrites (0.5-l pm in diameter) of labeled neurons also contained occasional LCV (60-100 nm) heavily labeled with peroxidase reaction product (Fig. 5A). ENK-IR neurons in the VMH received synaptic inputs
FIG. 6. Electron micrographs of ENK-IR axon terminals which are in synaptic contact with unlabeled dendrites in the VMH, lmmunoprecipitate is seen prominently in the LCV (A,C,D). and through the axoplasma. Smail clear vesicles are packed in ENK-IR terminals, particularly prominent in a bouton shown in (Bf. These axons frequently make synaptic contacts with unlabeled dendritic shafts (A,B) and dendritic spines (C,D). A typical citemal oganelle is seen at the neck of a spine (C). Most of these contacts are asymmetrical (A.C,D). Double smalf arrows show postjunctiooai dense body (D). Scales: 0. I pm.
from unlabeled boutons on their somata (Figs. 3, 4) and dendrites (Fig. 5). These unlabeled boutons contained many small, clear vesicles 30-50 nm in diameter (Figs, 3B, 4B,4C, 5), which were round, oval, or flattened with or without occasional LCV. The unlabeied axonal boutons very frequently made synaptic contacts with ENK-IR dendrites (Fig. 5), and less frequently with soma of ENK-IR neurons (Figs. 3B, 4B, 4C). Axo-dendritic synapses were most frequentIy asymmetrical, while axo-somatic synapses were primarily symmetrical.
IiNK-IA
Axons
Light ~is~~s~~~~~. A dense plexus of ENK-IR fibers were distributed in the VMH of untreated animds throughout the entire rostrocaudal and dorsoventral areas. The location, seen by light microscope, of ENK-IR fibers in the VMH was described in detail elsewhere [4, 23, 243. Elrctrtm microscopy. ENK-IR fibers were generally unmyelinated, axons (0.1-0.5 wrn in diameter) and axonal boutons (0.4-1.2 pm in diameter) were seen throughout the
165
ELECTRON MICROSCOPY OF VMH ENK NEURGNS
FIG. 7. Electron micrographs of an unlabeled VMN neuron which is in symmetrical synaptic contact with BNK-IR axonal boutons (thick and thin arrows). (B) is a high power view of a part of(A) (thick arrows). Asterisksshow ENK-IRaxonalboutons (B). Scales:(A) 1pm; (B) 0.1 pm.
VMH. These axonal boutons contained many small, clear vesicles (30-50 nm) and occasional LCV in which a heavy precipitate of peroxidase reaction product was visible (Fig. 6). Immunopr~ipi~te was diffused th~~~out the axeplasma, and was seen on the membranes of cell organelles. ENK-IR +xonal boutons formed synapses with dendritic shafts (Fig. 6 A and B), dendritic spines (Fig. 6 C and D), aad less frequently with the soma of unlabeled ne~ons (Fig. 7). Most of the neurons postsynaptic to ENK-IR axons were IO-25 Frn along the long axis and had a relatively sparse ~p~tion of LCV (60-230 nm with a predominance in the 6os(o run). No mtied differences were found between these neurons and ENK-IR neurons in the VMH. Axo-somatic synapses were mostly symmetrical (Fig. 7B) and axodendritic synapses were most frequently asymmetrical (Fig. 6 A, C, D). Labeled boutons were occasionally in apposition to the labeled neuronal soma and dendrites but rarely made axe-somatic synapses (data not shown).
DISCUSSION The present fmdings showed that the rat VMH contained many ENK-IR neurons. Dense plexuses of ~muno~ve fibers in the VMH formed synapses with various neuronal elements. The Golgi method has shown two kinds of neurons in the VMH [17]. Most are Type I cells with soma 10-30 pm across and 2-3 principal dendrites. The Type II cells seem to make up no more than 14% of the neuronal population; they were mostly in the lateral part of the VMH, and had larger soma 30-50 w in diameter. bud 1171has also divided VMH neurons into two classes on ultrastructural observacells.” Most are the tions, “common” and “aeon “common cells” that contain short segments of rER, rosettes of unbound ribosomes, and chromatoid bodies. They frequently receive synaptic inputs on the soma. The “anco~on cells” are larger with more knot rER and Nissl bodies. The “common cds” seem to correspond with
Type I cells and the “uncommon cells” with Type II [17]. Our findings have suggested that ENK-IR neurons were the “common cells,” since ENK-IR neurons are 10-25 pm across, and have single, short strands of rER, rosettes of unbound ribosomes, a few small Nissl bodies, and frequent synaptic contacts on the soma. Light microscopic observation of ENK-IR cells suggested that ENK-IR neurons and Type II cells were more or less in the same place, in the ventrolateral portion of the VMH. However, ultrastructural features of ENK-IR neurons corresponded to the “common cells,” which seem to be Type I cells by Golgi impregnation. Yamano rr ul. [23] found, using immunohistochemistry combined with knife cuts, electric lesion, and retrograde tracers, that enkephalinergic fibers in the VMH largely originate from the paraventricular hypothalamic nucleus. ENK-IR fibers in the VMH markedly decrease in most areas of the VMH after a lesion is made in the paraventricular nucleus, while most fibers in the ventrolateral part remain intact [23]. Thus, most ENK-IR fibers in the VMH except the ventrolateral area, arise from paraventricular ENK-IR neurons, while most fibers in the ventrolateral part of the VMH probably arise from locally synapsing axons belonging to ENK-IR neurons in the VMH. These fibers formed synaptic contacts with the dendrites and soma of VMH neurons. Perhaps the effect of opioids on the neuronal activity of VMH neurons [2,9], including glucoreceptor VMH neurons [18], is regulated by synapsing axons originating from ENK-IR paraventricular neurons or intrinsic VMH neurons. Ono er ~1. [18l showed that the direct excitatory &&%f
[ 121. Both of these manipulations interfere with enkephalin, which is perhaps serving as an excitatory transmitter or neuromodulator, mimics the excitatory effect of glucose in the VMH [ 191, so our results are in exact agreement with theirs. ENK-IR neurons scattered in the dorsal part of the VMH give rise to axons that innervate the midbrain central gray matter [25]. The midbrain central gray matter, where dense ENK-IR fibers are seen, is known to be a component of an opioid-mediated pain-suppression system 1131. Thus, ENK-IR neurons projecting to the midbrain central gray matter may modulate the neuronal activity in this area. in association with an analgesic system. While the VMH rcceives major inputs from the amygdala, the parabrachial nucleus [14], and the rostra1 hypothalamus. including the preoptic area [22]. Various neuropeptides are contained in these afferent systems. For example, neurotensin is found in neuronal pathways from the amygdala to the VMH 14.71. cholecystokinin in parabrachial-VMH pathways (4, 6, 261, and ENK in paraventriculo-VMH pathways 14.231. Thus, these neuropeptides in the VMH may be involved in regulation of various VMH functions such as feeding [I. I& 121, motivation [16], analgesia [13,25], and reproduction [8] in relation to the neuronal activity in the various brain areas.
ACKNOWLEDGEMENTS
The authors wish to thank Dr. Y. Shiotani (Department of Neuroanatomy, Institute of Higher Nervous Activity, Osaka University Medical School, Osaka, Japan) and Dr. T. Ono (Department of Physiology, Faculty of Medicine, Toyama Medical and Pharmaceutical University) for the valuable and critical comments. This work was supported by Grant-in-Aid for Scientific Research from the Japanese Ministry of Education.
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