Brain Research, 274 (1983) 17-23 Elsevier
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Leucine-Enkephalin-Containing Neuron System in the Facial Nucleus of the Rat with Special Reference to its Fine Structure EMIKO SENBA and MASAYA TOHYAMA Department of Neuroanatomy, Institute of Higher Nervous Activity, Osaka University Medical School, 4-3-57 Nakanoshima, Kitaku Osaka 530 (Japan) (Accepted January 18th, 1983) Key words: enkephalin - - 6-receptor - - motor function - - facial nucleus - - fine structure - - axo-dendritic contact
The light and electron microscopic localization of leucine-enkephalin-containing terminals in the facial nucleus of the rat were investigated by means of the peroxidase-antiperoxidase (PAP) immunocytochemical technique. By light microscopy, leucine-enkephalin-like immunoreactive (LEI) terminals were unevenly distributed in the facial nucleus. The greatest accumulation of the terminals was seen in the medial part of the nucleus. Electron microscopic examination of LEI-terminals in the medial part of the nucleus revealed that the predominant type of synaptic contacts of LEI-terminals in this area were axo-dendritic contacts (about 75%). These dendrites which made synapses with LEI-terminals were relatively large and rich in cytoplasmic organella, suggesting that they belonged to the proximal segment of the dendrite. A small number of LEI-terminals was found to make synaptic contact with neuronal perikarya (5%). These perikarya were very large and had nuclei with less chromatin particles. These findings suggest that LEI-terminals make contact with neurons which exist in the facial nucleus. The rest of the LEI-terminals (20%) were in apposition to the non-labelled axon terminals which contain small, clear and round vesicles. INTRODUCTION Enkephalins are endogenous opioid peptides which have a strong affinity for opioid receptors ~6, and they are believed to play an important role in the pain transmission system 15. In the dorsal horn of the spinal cord, they are supposed to have presynapticlS.19.22 and postsynaptic 36 inhibitory effects on the primary afferent substance P fibers. However, subsequent immunohistochemical studies have revealed the widespread distribution of enkephalins and suggested multiple functional roles of these peptides 21-29.34. For example, in addition to sensory areas, the motor conducting system, such as the cranial motor nuclei and ventral horn of the spinal cord, contain many enkephalin-immunoreactive terminalslO.13.14.26.32. Furthermore, recent pharmacological studies have shown that there exist multiple opiate receptors 20, and that enkephalin (6) receptors are related to epileptogenesis and other behavioural responses 4.11.30.33. The distribution of morphine (~) and enkephalin (6) receptors within the central nervous 0006-8993/83/$03.00 © 1983 Elsevier Science Publishers B.V.
system has been investigatedS, and it is said that, in the spinal cord, the ratio of 6-receptors to/~-receptors is much higher in the ventral horn compared to the dorsal hornt2. On the other hand, a recent physiological study has suggested that enkephalins might have excitatory effects on spinal motoneurons 3. These findings strongly suggest that enkephalins play an important role in the motor conducting system, besides their role in the pain transmission system. In fact, it has been shown that the areas related to motor functions contained a number of enkephalincontaining terminals. Recently, we have demonstrated that ieucine-enkephalin (LE) terminals located in the facial nucleus (FN) is innervated by LEcontaining cells in the caudal medullary reticular formation28, suggesting that this system is involved in the motor function of the FN. However, in order to explore the function of L E in the FN, it would seem to be important to elucidate the ultrastructures of LE fibers and the postsynaptic elements with which they make contact in the FN. In the present study, therefore, we attempted to describe the ultrastructural profiles of L E fibers in the FN.
18 MATERIALS AND METHODS
Leucine-enkephalin (LE) antibodies were raised in rabbits using 1-ethyl-(3,3-dimethylaminopropyl)carbodiimide (CDI) to conjugate the synthetic LE to bovine thyroglobulin (Sigma) as previously described by Elde et a1.10 The cross-reactivity of the antiserum and control experiments have already been described in our previous report 27.
The stained sections were briefly washed in PBS, postfixed for 1 h with 1% glutaraldehyde in PBS and treated with 1% OsO4 in PBS for 2 h at 4 °C. The tissue was dehydrated with graded alcohols and embedded flat in Epon. Serial ultrathin sections were taken and counterstained with uranylacetate and lead citrate before viewing on a 100-CX (JEL) electron microscope. Terminology of subnuclei of the FN is based upon the nomenclature of Papez 23.
Animals and tissue preparation
RESU LTS
Preparation of antiserum
Adult, male, albino rats (100-150 g body weight), anesthetized with pentobarbital, were perfused transcardially with t00 ml of saline, followed by 500 ml of ice-cold modified PLP fixative 17 containing 4% paraformaldehyde in 0.04 M phosphate buffer with sodium periodate (0.55 g/I) and lysine (3.42 g/l). After that the lower brainstem was removed, cut into 5 mm-thick blocks and immersed in fresh fixative overnight at 4 °C. After washing in 0.1 M phosphate buffer containing 7% sucrose for 12-24 h, sections were cut with a vibratome (25-30 ~m) and then subsequenty processed using immunocytochemical techniques.
Light microscopic observations As shown in Fig. la, numerous LE-immunoreactive (LEI) fibers were found in the facial nucleus (FN). It should be stressed that these LEI fibers were heavily concentrated in the medial subnucleus and a moderate number of fibers could be observed in the dorsal part of the nucleus. Only a few fibers could be observed in other areas of the nucleus. Fig. lb shows punctate varicosities and intervaricose fibers which appeared to be surrounding neuronal perikarya in the medial subnucleus of the facial nucleus. Each varicose fiber showed a great variety in the sizes of its punctate varicosities.
Immunocytochemistry The sections were incubated at room temperature in 20% dimethylsulfoxide in phosphate-buffered saline (PBS) for 1 h to promote the penetration of antibodies through the membrane 35, and then in 10% normal goat serum (NGS) in PBS for 2 h to prevent non-specific reactions. After the sections had been washed in PBS for 30 min, they were incubated with anti-LE antiserum diluted 1:1000 at room temperature overnight and washed in ice-cold PBS (30 rain x2). Then they were incubated in goat anti-rabbit IgG (Behring) diluted 1:50 at room temperature for 2 h, washed as before and incubated in rabbit peroxidase-anti-peroxidase antiserum ( D A K O ) diluted 1:50 at room temperature for 2 h. All antibodies were diluted in PBS containing 0.1% NGS. Then these sections were rewashed in ice-cold PBS for a further 30 min, transferred to Tris-HCl buffer (0.05 M, pH 7.4) containing 0.04% DAB for 30 min, then reacted with DAB and hydrogen peroxide in the same buffer for 10 min. Several sections were taken for light microscopy.
Electron microscopic observations General details of LEl-fibers Because LEI-fibers were concentrated in the medial subnucleus of the FN as described above, ultrastructural observations were performed mainly on this area. The labelled terminals were easily identified under the electron microscope, because of the dense precipitations in the LEI-terminals. The diameter of these terminals was 0.6-1.24.tm (mean 0.88/~m). In the labelled axon terminals, immunoreactive products were found within the large vesicles (about 80-100 nm in diameter) (Fig. lc), while the small vesicles (about 40-50 nm in diameter) were usually devoid of immunoprecipitates (Figs. lc and 2d). However, in the sections taken from the superficial layer of the embedded sections, reaction products were often associated on the surface of small vesicles and mi° tochondria in addition to large vesicles. Furthermore, it should be noted that there were fewer LEcontaining large vesicles than small clear vesicles and they were located far from the contact membrane.
19 belled neuronal components were analyzed. For this analysis, more than 150 micrographs of well-preserved LEI-terminals photographed at random were characterized.
Fig. 1. a and h: light photomicrographs showing punctate varicosities of LEI-terminals in the facial nucleus (FN). Note that LEI-terminals are concentrated in the medial suhnucleus (m) and dorsal part of the FN. A small number of varicosities are observed in the surrounding reticular formation, especially in the n. reticularis magnocellularis (rmg). In a higher magnification of the medial subnucleus, numerous varicose fibers are seen surrounding unlabelled neuronal perikarya (Fig. lb). c: electron microscopic photograph showing two LEI-terminais (E 1 and E2). Note that LE-positive immunoprecipitates are confined to large granular vesicles and small vesicles are devoid of reactions (El). a, x55; b, ×220; c, x 19,000.
Synaptic organization Various relationships between labelled and unla-
Axo-dendritic contact The predominant type of synaptic contact of LEIterminals observed in the facial nucleus were axodendritic synapses which accounted for up to 75% of all the characterized synaptic contacts. These contacts had a conventional kind of symmetrical synapse; that is, at the contact zone, such features as the following were observed: (1) pre- and post-synaptic membranous specialization (Fig. 2a, c and d); (2) presynaptic duster of synaptic vesicles close to the presynaptic membrane (Figs. 2b, d and 4a); (3) intersynaptic filaments (Fig. 3); and (4) widening of the intercellular gap. The dendrites which made contact with LEI-terminals in the FN were relatively large in diameter and measured 0.6-3.2 p m (mean 1.29 pro). They often contained some mitochondria and numerous cytoplasmic organelles such as rough endoplasmic reticulum and free ribosomes (Fig. 2c). It should be noted that in some cases a part of the smooth endoplasmic reticulum was situated close to the postsynaptic membrane like a subsynaptic cistern (Fig. 2a). In addition to symmetrical synapses, LEI-terminals in a few instances formed asymmetrical synapses with dendrites. However, in these cases, the diameter of the dendrites was relatively small (mean 0.85 ~m) and lacked cytoplasmic organelles. The dendrites which made contact with LEI-terminals were also surrounded by several non-labelled axon terminals and made symmetrical and asymmetrical synaptic contacts (Fig. 2a-d). Large dendrites seemed to make synaptic contacts with more axon terminals than small ones did, and some of them showed a rosette-like appearance. Most of these surrounding terminals, apart from the LEI-terminals, contained only small round clear vesicles, but in rare cases a few non-labelled large granular vesicles were observed in these axon terminals. Axo-axonic contact About 20% of the LEI-terminals located in the FN were in direct apposition to non-labelled axon terminals. At the contaet zone, although intersynaptic filaments were observed, the accumulation of dense
20
Fig. 2. Electron microscopic photographs showing axo-dendritic contacts, a and b: photographs taken from serial sections. The dendrite (D) which makes contact with the LEI-terminai (E) is relatively large in diameter and makes synaptic contact with at least two other axon terminals. At the site of synaptic contact with LEI-terminals, prominent synaptic membrane specializations and subsynaptic cisterns are observed (indicated by an arrowhead in Fig. la), c: LEI-terminal (E) makes synaptic contact with a dendrite of large diameter (2.4 ~m) which contains some endoplasmic reticulum (ER) (indicated by an arrow), d: LEI-terminal (E) makes synaptic contact with a rather small dendrite (D). a, × 18,000; b, × 18,000; c, × 15,000; d, x 16,600.
m a t e r i a l s on the c y t o p l a s m i c face o f b o t h m e m b r a n e s
in Fig. 3, L E I - t e r m i n a l s w e r e in a p p o s i t i o n to non-la-
and w i d e n i n g of t h e i n t e r c e l l u l a r gap w e r e n o t so c o n -
belled t e r m i n a l s in which small, r o u n d and clear synaptic vesicles w e r e p a c k e d . It was v e r y difficult to
spicuous as those of a x o - d e n d r i t i c contacts. A s s h o w n
21 terminal and the other is from the unlabelled one. Furthermore, it should be noted that these perikarya which make contact with LEI-terminals, had a large nucleus with less chromatin particles and a prominent nucleolus, surrounded by an ample cytoplasmic area which contained abundant rough endoplasmic reticulum, free ribosomes and mitochondria. DISCUSSION
Fig. 3. Electron microscopic photographs showing axo-axonal relationships, a and b: photographs taken from serial sections. LEI-terminal (E) is in apposition to non-labelled axon terminal (A) which contains numerous small clear vesicles, a, x 18,000; b, x 18,000. identify which element belonged to the presynaptic group in structure in spite of observations of many serial sections, though in some cases synaptic vesicles located in the LEI-terminals and in rare instances synaptic vesicles in the non-labelled terminals, tended to accumulate close to the membrane which was facing the non-labelled terminals or LEI-terminals, respectively. Axo-somatic contact
In rare cases (about 5%), LEI-terminals in the FN were found to have a symmetrical synapse with neuronal perikarya (Fig. 4). At the contact zone, an accumulation of synaptic vesicles, dense materials associated with the membrane of LEI-terminals, and intersynaptic filaments were observed. Fig. 4 demonstrates that a single cell somata receives at least two types of afferent impulses; one is from the labelled
The present study has shown that LEI-terminals are concentrated in the medial and dorsal parts of the FN. Somatotopic organization of the FN was investigated on various kinds of animals, including rats, by means of degeneration6,23, 31 or H R P injection 25 methods, and it has been clearly shown that neurons of these areas of the nucleus innervate auricular muscles. These findings strongly suggest that enkephalins might regulate the movements of the facial muscles, especially those of auricular muscles. As to the function of enkephalins in the FN, further phylogenetical and physiological studies will be inevitable. Recently our immunohistochemical study showed that LEI-terminals located in the FN originate from LEI-neurons located in the caudal medullary reticular formation 2s. The present study has demonstrated that the axon terminals from these cells make synaptic contact mainly with large dendrites and in a few cases with cell somata. As is clearly demonstrated in this study, the dendrites which make synapses with LEI-terminals in the FN are relatively large and rich in cytoplasmic organelles, so these dendrites seem to belong to the proximal segment of the dendrite. Thus it is likely that these dendrites originate from the neurons located within the FN. In addition, the present study has also revealed the direct synaptic contact of LEI-terminals with the neurons located in the FN. Hence it could be concluded that LEI-terminals directly influence the neurons or proximal segments of them via axo-somatic or axo-dendritic contacts, though it is not clear in this study whether these cells belong to motoneurons or interneurons in the FN. The present study has also shown that a substantial number of LEI-terminals are in direct apposition to non-labelled axon terminals. Although clear evidence that they make synaptic contacts could not be obtained in this study, it still remains a possibility that there exists direct neural transmission between these
22
Fig. 4. Electron microscopic photographs showing axo-somatic contacts, a and b: photographs taken from serial sections. LEI-terminal (E) makes symmetric synaptic contact with neuronal perikarya, which contain abundant mitochondria (M), rough endoplasmic reticulum (ER) and free ribosomes (r). Note the dense materials and accumulation of synaptic vesicles at the presynaptic membrane (indicated by an arrow), a, x20,000; b, x20,000. terminals and that they influence the function of the FN concomitantly. The paucity of pre- and post-synaptic membranous specialization might be accounted by a rather weak tissue fixative employed in this study compared to ordinary fixatives for electron microscopy. As to the origins of these non-labelled axon terminals facing the LEI terminals, the following sites could be mentioned as possible origins: the red nucleus, the cuneiform nucleus, the spinal trigeminal nucleus, etc., because we could observe HRP-labelled cells in these areas following H R P injection into the FN (unpublished observations). In fact, rubrobuibar fibers to the FN have been advocated by Courville 7 by means of the degeneration-silver impregnation method and descending projection from the cuneiform nucleus to the facial nucleus has been REFERENCES 1 Aronin, N., DiFiglia, M., Liotta, S. and Martin, J. B., U1trastructural localization and biochemical features of immunoreactive Leu-enkephalin in monkey dorsal horn, J.
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