β-endorphin-, adrenocorticotrophic hormone- and neuropeptide y-containing projection fibers from the arcuate hypothalamic nucleus make synaptic contacts on to nucleus preopticus medianus neurons projecting to the paraventricular hypothalamic nucleus in the rat

Arcuate POMC and NPY input to PVN-projecting POMe neurons Neuroscience Vol. 98, No. 3, pp. 555–565, 2000 555

Pergamon PII: S0306-4522(00)00134-2

䉷 2000 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522/00 $20.00+0.00

www.elsevier.com/locate/neuroscience

b-ENDORPHIN-, ADRENOCORTICOTROPHIC HORMONE- AND NEUROPEPTIDE Y-CONTAINING PROJECTION FIBERS FROM THE ARCUATE HYPOTHALAMIC NUCLEUS MAKE SYNAPTIC CONTACTS ON TO NUCLEUS PREOPTICUS MEDIANUS NEURONS PROJECTING TO THE PARAVENTRICULAR HYPOTHALAMIC NUCLEUS IN THE RAT H. KAWANO* and S. MASUKO Department of Anatomy, Saga Medical School, Nabeshima 5-1-1, Saga, 849-8501, Japan

Abstract—The nucleus preopticus medianus is known to be situated in a key site in pathways regulating the paraventricular hypothalamic nucleus. To investigate the innervation pattern to nucleus preopticus medianus neurons by afferent fibers containing b-endorphin, adrenocorticotrophic hormone and neuropeptide Y, a retrograde tracing method was combined with immunohistochemistry for these peptides in the rat. In the first experiment with injection of a retrograde tracer in the nucleus preopticus medianus, retrogradely labeled neurons were found in many regions throughout the brain. Among these, the arcuate hypothalamic nucleus contained a number of retrogradely labeled neurons showing immunoreactivity to the neuropeptides examined. About 20%, 20% and 40% of retrogradely labeled arcuate hypothalamic nucleus neurons showed b-endorphin, adrenocorticotrophic hormone and neuropeptide Y immunoreactivity, respectively. About 18% and 57% of retrogradely labeled neurons in the nucleus tractus solitarius and ventrolateral medulla, respectively, were immunoreactive to neuropeptide Y. There were many more neuropeptide Yimmunoreactive projections to the nucleus preopticus medianus from the arcuate hypothalamic nucleus than those from the medulla. None of the retrogradely labeled neurons in the medulla showed immunoreactivity to b-endorphin or adrenocorticotrophic hormone. In the second experiment with injection of a retrograde tracer in the paraventricular hypothalamic nucleus, electron microscopic observation revealed that retrogradely labeled neurons in the nucleus preopticus medianus were in synaptic contact with b-endorphin-, adrenocorticotrophic hormone- and neuropeptide Y-immunoreactive axon terminals. The present finding indicates that nucleus preopticus medianus neurons projecting to the paraventricular hypothalamic nucleus are innervated by b-endorphin-, adrenocorticotrophic hormone- and neuropeptide Y-containing arcuate hypothalamic nucleus neurons in addition to being innervated by neuropeptide Y-containing catecholaminergic medullary neurons which have been reported in our previous study. 䉷 2000 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: proopiomelanocortin-derived peptides, retrograde tracing, immunohistochemistry, electron microscopy, nucleus tractus solitarius, ventrolateral medulla.

A variety of effects of bEND and ACTH has been reported, including analgesic effect, hypotensive and hypertensive effects, water intake and vasopressin (VP) release. 8,15,35,46,49,50 The hypertensive effect of intracerebroventricular application of bEND has been reported to be abolished in rats neonatally treated with monosodium glutamate, 50 which selectively destroys structures around the third ventricle, including the ARC. 36 Hence, the ARC, as a production site of POMCderived peptides, is important for cardiovascular function. It has also been demonstrated that the ARC contains a number of neurons immunoreactive to neuropeptide Y (NPY), 9,11 which also influences VP secretion and the cardiovascular system. 39 In the POMe, nerve terminals immunoreactive to bEND, ACTH and NPY have been found. 2,4,9,17,21,22 Electrophysiological and morphological investigations have revealed the projection from the POMe to the PVN, 19,22,25,33,40,44 a storehouse of neurons containing VP whose hypotensive and antidiuretic roles are well known. Neuronal somata immunoreactive to POMC-derived peptides were later found in the nucleus tractus solitarius (NTS) as well. 18,27,42 The NTS, together with the ventrolateral medulla, has also been shown to be implicated in cardiovascular function and vasopressin release, 3,7,14,28,31,34 and both medullary regions contain NPY-immunoreactive neuronal somata. 9,11 To solve the mechanism controlling the autonomic functions, it is important to study the neuronal pathways among structures responsible for cardiovascular and body

A lamina terminalis-associated structure, the nucleus preopticus medianus (POMe), is one of the anteroventral third ventricle (AV3V) regions constituting the rostral wall of the third ventricle 33 and plays important roles in cardiovascular and body fluid homeostasis. 5,13,19,32,33 It has been demonstrated that in water-deprived and hypertonic saline-ingested rats, protein synthesis is increased in some specified regions, such as the POMe, subfornical organ (SFO), magnocellular parts of the paraventricular hypothalamic nucleus (PVN) and arcuate hypothalamic nucleus (ARC). 29 This suggests that these structures are also involved in body fluid regulation. In earlier studies the ARC has been shown as the only nucleus that contains neuronal somata immunoreactive to proopiomelanocortin (POMC)-derived peptides, such as b-endorphin (bEND) and adrenocorticotrophic hormone (ACTH). 1,17,44,48 *To whom correspondence should be addressed. Tel.: ⫹ 81-952-34-2223; fax: ⫹ 81-952-34-2015. E-mail address: [email protected] (H. Kawano). Abbreviations: ACTH, adrenocorticotrophic hormone; ARC, arcuate hypothalamic nucleus; AV3V, anteroventral third ventricle; bEND, bendorphin; DAB, diaminobenzidine-tetrahydrochloride; HEPES, N-2hydroxyethylpiperazine-N 0 -2-ethanesulfonic acid; IgG, immunoglobulin G; NPY, neuropeptide Y; NTS, nucleus tractus solitarius; PAP, peroxidase anti-peroxidase; PBS, phosphate-buffered saline; POMC, proopiomelanocortin; POMe, nucleus preopticus medianus; PVN, paraventricular hypothalamic nucleus; SFO, subfornical organ; VP, vasopressin; WGA–HRP–gold, wheat germ agglutinin–conjugated horseradish peroxidase–colloidal gold complex. 555

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fluid homeostasis in the hypothalamus and medulla oblongata with reference to the neurochemical substances that are involved. In the present study, we investigated immunohistochemically the origins of nerve terminals immunoreactive to bEND, ACTH and NPY in the POMe, after injection of a retrograde tracer into the POMe. Furthermore, the possibility of synaptic contacts of axon terminals immunoreactive to bEND, ACTH and NPY on to POMe neurons projecting to the PVN was also clarified under electron microscopy, after injection of a tracer into the PVN. The existence of synaptic inputs of NPY-immunoreactive axon terminals on to PVNprojecting POMe neurons has been previously demonstrated. 22 Parts of the present study have been published in abstract form. 23,24,26 EXPERIMENTAL PROCEDURES

Male Sprague–Dawley rats (49 ^ 1 days old) were used. Animals were kept under controlled light/dark conditions with food and water available ad libitum. All animal experiments were approved by the Saga Medical School Animal Care and Use Committee. Before each operation and perfusion fixation, animals were anesthetized with intraperitoneal injection of pentobarbital sodium (50 mg/kg body weight). The present study was comprised of two experiments, to detect origins of nerve terminals immunoreactive to POMC-derived peptides and NPY in the POMe (Experiment 1) and to detect synaptic contacts of axon terminals immunoreactive to these neuropeptides on to POMe neurons projecting to the PVN (Experiment 2). Experiment 1 A small quantity (0.3 ml) of a retrograde tracer, wheat germ agglutinin–conjugated horseradish peroxidase–colloidal gold complex (WGA–HRP–gold), was stereotaxically injected into the POMe from the dorsal aspect, using a glass needle with an outer diameter of about 50 mm (n ˆ 8). 25 The tip of the needle was fixed to the skull with a self-curing resin, MEND-REX (Nissin Dental Products, Kyoto, Japan). 22 After two to five days, 100 mg colchicine in 5 ml physiological saline was administrated into the lateral ventricle at a speed of 1 ml/min. On the following day (over 24 h), animals were fixed by transaortic perfusion of physiological saline followed by 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.3). The brain was divided into three pieces by cutting at the hypothalamo-midbrain and pons-medulla junctions and re-fixed by immersion in the same fixative for 2–24 h. After cryoprotect treatment in 20% sucrose in phosphatebuffered saline (PBS) overnight, free-floating 40-mm-thick frontal sections from each brain block were serially cut by a freezing microtome, and collected in order in 12 PBS-containing-wells of a microtiter plate. The sections in a well were mounted on gelatin-coated glass slides and counterstained by hematoxylin to examine the injection site (POMe sections) and to define the nucleus demarcation (sections of other levels) without any treatment. The sections in each of the remaining wells were processed for further silver intensification and immunohistochemistry. Animals in which the tracer did not reach the POMe but was deposited in the medial septum were used as controls (n ˆ 3). Experiment 2 A small quantity (0.3 ml) of WGA–HRP–gold was stereotaxically injected into the PVN from the dorsal aspect (n ˆ 3). 22,25 After two to seven days, the animals were fixed and sections containing the PVN were obtained as described above and mounted on gelatin-coated glass slides. When the tracer was centered in the PVN, sections containing the POMe were serially cut at a thickness of 20 mm throughout the entire rostrocaudal extent of the nucleus by a microslicer (D. S. K., Kyoto, Japan) and processed for further silver intensification and immunohistochemistry. Silver intensification and immunohistochemistry Free-floating sections throughout the entire brain (Experiment 1) and of the POMe (Experiment 2) were incubated with silver developer for 7 min and fixed in 2.5% sodium thiosulfate in 0.02 M HEPES buffer (pH 6.8). 22 After that, the silver intensified sections were soaked

in 0.3% H2O2 in PBS to block activities of intrinsic and retrogradely transported peroxidase, rinsed in PBS, and preincubated with 2% normal swine serum in PBS. The preincubation medium was also used for diluents of all antisera employed. The sections were then incubated with polyclonal rabbit anti-bEND (Peninsula Laboratories, CA, U.S.A., Lot: 010165-3; diluted 1:4000), polyclonal rabbit anti-ACTH (Peninsula Laboratories, CA, U.S.A.; Lot: 021080-1; diluted 1:1000) or monoclonal mouse anti-ACTH (Novocastra Laboratories, Benton Lane, U.K.; Lot: 0802; diluted 1:150), or polyclonal anti-NPY (Peninsula Laboratories; Lot: 031215-6; diluted 1:5) overnight at 4⬚C. The sections were subsequently incubated with unlabeled swine anti-rabbit IgG (DAKOPATTS, Glostrup, Denmark; diluted 1:40) or unlabeled swine anti-mouse immunoglobulin (IgG) (Nordic Immunological Laboratories, Tiburg, The Netherlands; diluted 1:50) and rabbit peroxidase anti-peroxidase (PAP) complex (DAKOPATTS; diluted 1:80) or mouse PAP complex (DAKOPATTS; diluted 1:80) for 1.5–2 h each at room temperature. Finally, the sections were incubated with 0.02% diaminobenzidine-tetrahydrochloride (DAB; Dotite, Kumamoto, Japan) in Tris-buffered saline (pH 7.6) containing 0.005% H2O2 for 10–30 min. The silver intensified and immunostained sections throughout the brain (Experiment 1) were mounted on gelatin-coated glass slides, dried and covered. Numbers of retrogradely labeled neurons from five rats were counted in silver intensified sections under dark illumination, and those of double-labeled as well as retrogradely labeled neurons were counted in silver intensified and immunostained sections under a light microscope. For electron microscopic observation, the silver intensified and immunostained sections containing the POMe (Experiment 2) were dipped in 0.05% tetrachloroauric acid in 0.1 M phosphate buffer (pH 7.3) to tone up the retrograde labeling and DAB reaction products, postfixed with 1% osmium tetroxide and 1.5% potassium ferrocyanide in 0.1 M phosphate buffer, dehydrated, and embedded in Spurr’s resin (Nisshin EM, Tokyo, Japan). Ultrathin sections of the POMe were prepared on a Sorvall MT2-B Ultra Microtome, and observed under a JEOL 100CX electron microscope without metal staining. An additional two rats without any treatment were employed to examine the localization of bEND-, ACTH- and NPY-immunoreactive nerve terminals in the POMe. Animals were fixed by perfusion of acetic acid-free Bouin’s solution and 5-mm-thick paraffin sections were cut. 21 Immunostaining procedures for each peptide were as described above.

RESULTS

In the intact animals, axon varicosities immunoreactive to bEND and ACTH were moderately to abundantly distributed throughout the POMe (Fig. 1). These immunoreactive axon varicosities showed, however, a regional difference in frequency; they were slightly more abundant in the ventral than the dorsal parts and at the middle to caudal than rostral levels. In the present study, bEND-immunoreactive axon varicosities were more frequently observed than ACTHimmunoreactive axon varicosities in the POMe (Fig. 1). More numerous axon varicosities immunoreactive to NPY than to POMC-derived peptides were also found throughout the POMe, particularly in the ventral parts of the nucleus, as shown in our previous report 21,22 (data not shown). In the medial septum, a structure dorsal to the POMe, however, bEND-, ACTH- and NPY-immunoreactive axon varicosities were quite sparse. In the experiments of retrograde tracing in the ARC from the POMe (Experiment 1), the injection site of WGA–HRP– gold was located in the dorsal, ventral or entire dorsoventral part as well as at the middle or the caudal level of the POMe (Fig. 2), although a little diffusion in the medial septum along the injection needle was seen in some cases. After these injections, retrogradely labeled neurons were found in several brain regions, such as the ARC, SFO, PVN, dorsomedial hypothalamic nucleus, midbrain central gray, lateral parabrachial nucleus, locus coeruleus, floor of the fourth ventricle (C3),

Arcuate POMC and NPY input to PVN-projecting POMe neurons

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Fig. 1. Serial frontal sections showing immunohistochemistry for b-endorphin (A) and adrenocorticotrophic hormone (B) of the nucleus preopticus medianus at a middle level (about 100 mm rostral to the crossing of the anterior commissure). Immunoreactive axon varicosities are richly seen in the nucleus preopticus medianus (indicated by a broken line), but few in the medial septum (MS). v, third ventricle. Scale bar ˆ 100 mm.

NTS and the ventrolateral medulla oblongata (Fig. 2), as has been previously reported. 10,25,30,37,38,45 Among the regions in which retrogradely labeled neurons and immunostained neurons appeared, double-labeled neurons were found in only three regions: the ARC, NTS and ventrolateral medulla. In the ARC, retrogradely labeled neurons were bilaterally distributed throughout the rostrocaudal extents of the nucleus (Fig. 2, Table 1) with a peak at the middle levels. There was little relationship between the rostrocaudal injection level and the distribution of retrogradely labeled neurons. They were mainly distributed within and somewhat dorsolateral to the ARC (corresponding to the site possessing group I of bEND-immunoreactive neurons which have been shown by Ref. 4). When the injection was located in the

dorsal part, retrogradely labeled neurons tended to occur in the region lateral to the ARC and almost ventrolateral border of the hypothalamus (corresponding to the site of group II neurons 4), as well (Fig. 2). Although the number of retrogradely labeled neurons in these ARC regions varied from rat to rat, probably due to unavoidable differences in the amount of tracer deposited, 40–100 neurons per section were retrogradely labeled in both sides (Table 1). Immunohistochemistry combined with retrograde labeling revealed that both bENDand ACTH-immunoreactive retrogradely labeled neurons were found in the regions corresponding to group I and II neurons, with a tendency to be located preferentially in the dorsolateral part (Figs 3, 4). About 20% of retrogradely labeled neurons in the ARC regions were estimated to be immunoreactive to bEND or ACTH (Table 1).

Table 1. Number of retrogradely labeled and/or immunoreactive neurons throughout the arcuate hypothalamic nucleus, nucleus tractus solitarius and ventrolateral medulla counted in sections of 480-mm intervals Animal

ARC Retrograde

747

199

752

392

755

482

756

456

757

202

Mean

346.2

Retrograde ⫹ END (%)* 52 (26.1%) 109 (27.8%) 59 (12.2%) 88 (19.3%) 51 (25.2%) 71.8 (20.7%)

Retrograde

348 409 410 433 291 378.2

NTS

Retrograde ⫹ ACTH (%) 52 (14.9%) 118 (28.9%) 65 (15.9%) 94 (21.7%) 56 (19.2%) 77.0 (20.4%)

Retrograde

426 551 479 453 294 440.6

Retrograde ⫹ NPY (%0) 168 (39.4%) 248 (45.0%) 117 (24.4%) 186 (41.1%) 163 (55.4%) 176.4 (40.0%)

Retrograde

31 14 4 12 4 15.3

*Percentages are represented by (number of double-labeled neurons)/(number of retrogradely labeled neurons).

VLM

Retrograde ⫹ NPY (%) 8 (25.8%) 2 (14.3%) 1 (25.0%) 1 (8.3%) 0 (0.0%) 2.8 (18.0%)

Retrograde

83 46 12 48 15 48.0

Retrograde ⫹ NPY (%) 51 (61.4%) 32 (69.6%) 1 (8.3%) 24 (50.0%) 2 (13.3%) 27.3 (56.8%)

558 H. Kawano and S. Masuko Fig. 2. Frontal sections showing injection sites of WGA–HRP–gold in the nucleus preopticus medianus (A, E, I, M), and retrogradely labeled neurons in the arcuate hypothalamic nucleus (B, F, J, N), nucleus tractus solitarius (C, G, K, O) and ventrolateral medulla (D, H, L, P) in four cases. Individual animal number is given in the upper right corner in A, E, I, M, and deposit of the tracer in the nucleus preopticus medianus is seen with (I) and without (A, E, M) silver intensification at levels about 40 mm (A, E) and 300 mm (I) rostral to, and just at (M) the crossing of the anterior commissure. Levels of the arcuate hypothalamic nucleus are about 800 mm (B), 650 mm (F), 850 mm (J), and 550 mm (N) caudal from the rostral pole of the nucleus. Retrogradely labeled neurons are brightly seen under darkfield illumination. Levels of the medulla are about 300 mm (C, D), 500 mm (G, H), 350 mm (K, L) and 550 mm (O, P) caudal to the obex. Arrowheads indicate ventral surface of the medulla. ac, anterior commissure; ap, area postrema; c, central canal; ts, solitary tract; v, third ventricle. Scale bars ˆ 200 mm (A; applies to E, I and M also), 100 mm (B; applies to C, D, F–H, J–L, N–P also).

Fig. 2 (M–P).

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Retrogradely labeled ARC neurons also exhibited immunoreactivity to NPY. The majority of double-labeled neurons was found within the nucleus and a few in the peripheral parts (Fig. 2). About 40% of retrogradely labeled neurons in the ARC regions were immunoreactive to NPY (Table 1). In control experiments, in which WGA–HRP–gold did not reach the POMe but was located in the medial septum, virtually no neurons were retrogradely labeled in the ARC and its lateral regions. In the NTS, bilateral retrogradely labeled neurons were mainly found in the medial subnucleus and occasionally in the intermediate and dorsolateral subnuclei 20 (Fig. 2). They were located at levels between 1 mm caudal to and 0.3 mm rostral to the obex with a peak (6–22 neurons/section) at the level of the area postrema. At these levels, NPY-immunoreactive neurons were scattered, as has been previously reported, 9,11 and NPY immunoreactivity was found in about 18% of retrogradely labeled neurons (Table 1). Although neurons immunoreactive to bEND and ACTH were also found in the caudal NTS, none showed simultaneous retrograde labeling (Fig. 5). In the ventrolateral medulla at levels between about 1.9 mm caudal to and 1.5 mm rostral to the obex, corresponding approximately to the caudal pole of the nucleus ambiguus, retrogradely labeled neurons were also bilaterally encountered (Fig. 2). They were predominantly distributed at the level of the area postrema (13–27 neurons/section). In the ventrolateral medulla, about 57% of retrogradely labeled neurons showed simultaneous NPY immunoreactivity (Fig. 5, Table 1). In the central gray, locus coeruleus and floor of the fourth ventricle, in which NPY-immunoreactive neurons have been demonstrated, 9,11 retrogradely labeled neurons were also intermingled with scattered NPY-immunoreactive neurons. However, no double-labeled neurons were detected in these regions. In the experiments of retrograde tracing in the POMe from the PVN (Experiment 2), each injection site of the WGA– HRP–gold was mainly confined within the magnocellular parts and diffused somewhat to the parvocellular parts of the PVN. After these injections, retrogradely labeled neurons were found in several regions in the brain, including the POMe, SFO, NTS and the ventrolateral medulla oblongata, as described in the series of our studies. 22,25 In the POMe, retrogradely labeled neurons were distributed throughout the rostrocaudal extent of the nucleus, especially in the ventral parts at the middle level just rostral to the crossing of the anterior commissure. Although the number of retrogradely labeled neurons varied according to the size of the injection, the POMe contained eight to 15 retrogradely labeled neurons/section on average, with a peak (15–20 neurons) at the middle level. Electron microscopic examination of the POMe revealed bEND, ACTH and NPY immunoreactivities in about 5% (ACTH) to 10% (NPY) of axon terminals. The morphological features of bEND-, ACTH- and NPY-immunoreactive axon terminals were generally similar: they were about 1 mm in diameter and were round, ovoid or ellipsoid in shape. Axoplasm of bEND- and ACTH-immunoreactive nerve terminals contained a substantial number of large cored vesicles and was packed with round, small clear vesicles. These axon terminals made more symmetric synapses (about 75%) than asymmetric synapses with dendrites (about 60%) and somata (about 38%) of neurons without immunohistochemical and

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Fig. 3. Adjacent sections of the arcuate hypothalamic nucleus, about 500 mm caudal from the rostral pole of the nucleus, showing double labeling for retrograde label and b-endorphin (A, B), adrenocorticotrophic hormone (C, D) and neuropeptide Y (E, F) after injection of WGA–HRP–gold in the nucleus preopticus medianus. Higher magnifications of parts of A, C and E are shown in B, D and F, respectively. Note that double-labeled neurons (arrows) and singly retrogradely labeled neurons (some of them are indicated by arrowheads) are intermingled with immunoreactive neurons. v, third ventricle. Scale bars ˆ 100 mm (A; applies to C and E also), 50 mm (B; applies to D and F also).

retrograde label. NPY-immunoreactive axon terminals contained many round small clear vesicles and some large cored vesicles, and formed more asymmetric synapses than symmetric synapses, as has been shown in our previous study. 22 Retrograde label was found in lysosomes of the dendrites and cell bodies of neurons in the POMe (Fig. 6). Some

immunoreactive axon terminals made synapses with neuronal elements containing retrograde label, in addition to those without retrograde label. Retrogradely labeled neuronal elements also received synaptic contacts from non-immunoreactive axon terminals (Fig. 6). More axo-somatic than axodendritic synapses and more symmetric than asymmetric

Arcuate POMC and NPY input to PVN-projecting POMe neurons

Fig. 4. A schematic drawing which shows the distribution of retrogradely labeled and double-labeled neurons in and adjacent to the arcuate hypothalamic nucleus after injection of WGA–HRP–gold in the nucleus preopticus medianus.

synapses were found between bEND- and ACTH-immunoreactive axon terminals and retrogradely labeled elements. In contrast, NPY-immunoreactive axon terminals made more axo-dendritic than axo-somatic synapses and more asymmetric than symmetric synapses on to retrogradely labeled elements (Fig. 6). DISCUSSION

In the present study, terminal varicosities immunoreactive to bEND and ACTH were found in the POMe. This is, in general, consistent with the previous finding. 2,4,17 Both peptides are derived from a common precursor, POMC, and undergo a processing to discrete peptides at perikaryon as well as terminal sites. Studies by double-labeling fluorescent immunohistochemistry have shown that these two peptides coexist nearly completely in single neurons in the

561

ARC. 6,27,43,47 The present electron microscopic observation revealed that there were no differences in morphological features and synaptic patterns between bEND- and ACTHimmunoreactive axon terminals in the POMe. However, we found more numerous terminal varicosities immunoreactive to bEND than to ACTH in the present study. This discrepancy concerning degrees of appearance of immunoreactive axon varicosities may be due to technical issues, especially the working properties of antibodies. We, therefore, consider that ACTH-immunoreactive axon varicosities in the POMe must be the same as bEND-immunoreactive axon varicosities. The present immunohistochemical study with injection of a retrograde tracer in the POMe revealed that retrogradely labeled bEND- and ACTH-immunoreactive neurons were uniquely found in the ARC, but not in the NTS, although in the latter nucleus, either retrogradely or immunohistochemically labeled neurons were found. This indicates that POMC-derived peptide-immunoreactive axon terminals in the POMe come exclusively from the ARC. However, retrogradely labeled NPY-immunoreactive neurons were found in the ARC, NTS and ventrolateral medulla. Medullary NPYimmunoreactive projections to the POMe were, in general, consistent with a previous finding. 10 A substantial number of NPY-immunoreactive neurons in the medulla oblongata has been shown to be catecholaminergic, 11 and catecholaminergic projections from the medulla to the POMe have also been demonstrated. 38,45 Accordingly, a greater part of NPY-immunoreactive projections from the medulla to the POMe appears to be catecholaminergic (mainly noradrenergic). 22 Total projections from the ventrolateral medulla were about threefold those from the NTS, while NPYimmunoreactive projections from the ventrolateral medulla in the present study were about tenfold those from the NTS, indicating that noradrenergic NPY-immunoreactive projections from the medulla to the POMe come predominantly from the ventrolateral medulla and to a lesser extent from the NTS. NPY-immunoreactive projections from the ARC were much more abundant than those from the ventrolateral medulla (about tenfold). Previous findings have shown that NPY, TH (dopaminergic) and POMC-derived peptide immunoreactivities are seen in separate populations of the ARC neurons. 6,11 Accordingly, the present study indicates that the POMe receives powerful inputs of non-catecholaminergic NPY-immunoreactive nerve terminals from the ARC, in addition to catecholaminergic NPY-immunoreactive projections from the medulla oblongata. We could not differentiate between these two NPY-immunoreactive axon terminals. This is a matter for further investigation. Our present and previous 22 studies revealed that axon terminals immunoreactive to bEND, ACTH and NPY were in synaptic contact with retrogradely labeled neurons in the POMe from the magnocellular part of the PVN. The present study provided the evidence that the ARC is the origin of POMC-derived peptide-immunoreactive axon terminals and an origin of NPY-immunoreactive axon terminals making synaptic contacts with POMe neurons projecting to the magnocellular parts of the PVN. Other destinations of ACTH- or bEND-immunoreactive neurons in the ARC have been reported: afferents to the midbrain central gray involved in analgesia 51 and to the periventricular nucleus of the hypothalamus involved in allowing somatostatin neurons there to release neurotransmitter. 12 ACTH- 41 and NPYimmunoreactive 16 projections from the ARC to the PVN

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Fig. 5. Sections of the caudal medulla showing double labeling for retrograde label and b-endorphin (A) and neuropeptide Y (B, C) after injection of WGA– HRP–gold in the nucleus preopticus medianus. Separate distribution of b-endorphin-immunoreactive neuron and retrogradely labeled neuron (arrowhead) in the nucleus tractus solitarius (A) and co-localization of neuropeptide Y immunoreactivity in retrogradely labeled neurons (arrows) in the nucleus tractus solitarius (B) and ventrolateral medulla (C) are seen at higher magnification in insets. Levels are about 700 mm (A), 40 mm (B) and 950 mm (C) caudal to the obex. c, central canal; ts, solitary tract. Scale bars ˆ 100 mm (A; applies to B also), 200 mm (C), 50 mm (insets).

have also been demonstrated, and these NPY-immunoreactive projections are suggested to be involved in controlling carbohydrate ingestion. 16 The NPY-immunoreactive neurons in the ARC may also give such information to POMe neurons which project to the PVN.

It has been shown that the POMe receives angiotensinergic inputs from the SFO 31 and corticotropin releasing factor and Leu-enkephalin projections from the lateral parabrachial nucleus. 30 Other neurochemical substances may also regulate POMe neurons, since we have previously demonstrated many

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Fig. 6. Electron micrographs of middle levels of the nucleus preopticus medianus showing synaptic contacts of peptide-immunoreactive axon terminals with retrogradely labeled neurons after injection of WGA–HRP–gold in the paraventricular hypothalamic nucleus. Axon terminals (asterisks) immunoreactive to b-endorphin (A), adrenocorticotrophic hormone (B) and neuropeptide Y (C) make synaptic contacts with cell bodies containing retrograde label within the lysosomes (arrows). Scale bars ˆ 0.5 mm.

nerve terminals immunoreactive to a variety of neuropeptides and monoamines, such as substance P, enkephalins and serotonin in the POMe. 21 Among such comprehensive innervation to the POMe, the present findings provided the evidence that POMe neurons projecting to the PVN are innervated by at least two different populations of ARC neurons

(NPY-immunoreactive neurons and ACTH/bEND-immunoreactive neurons) for regulation of critical autonomic functions, such as cardiovascular and body fluid homeostasis. Acknowledgements—The authors thank Ms Yuko Honda and Mr Toshimi Tabata for their technical assistance.

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