Development of Neuropeptide Y Innervation in the LiverWEI-GUANG DING,1 HIROSHI KITASATO,1 AND HIROSHI KIMURA2*1Department of Physiology, Shiga University of Medical Science, Otsu 520-21, Japan2Institute of Molecular Neurobiology, Shiga University of Medical Science, Otsu 520-21, Japan

KEY WORDS neuropeptide Y; development; liver; vertebrates; human

ABSTRACT Hepatic neuropeptide Y (NPY) innervation was studied by immunohistochemistryin various mature vertebrates including the eel, carp, bullfrog, turtle, chicken, mouse, rat, guineapig, dog, monkey, and human. In addition, an ontogenetic study on hepatic NPY was made indeveloping mice and guinea pigs. In all species examined except the eel, NPY-like immunoreactivitywas detected in nerve fibers. In the carp, bullfrog, turtle, chicken, mouse, and rat, NPY-positivefibers were distributed around the wall of hepatic vessels and the bile duct of the Glisson’s sheath.The density of NPY-positive fibers increased with evolution. However, in the guinea pig, dog,monkey, and human, numerous NPY-positive fibers were observed not only in the Glisson’s sheathbut also in the liver parenchyma. Positive fibers formed a dense network that surrounded thehepatocytes. The present immunoelectron microscopic study has confirmed that NPY-positiveterminals are closely apposed to hepatocytes. Ontogenically, NPY-positive fibers were first found inthe embryonic liver of 19-day-old mice. Positive fibers increased with age, and the highest peak wasseen 1 week after birth. However, NPY-positive nerve fibers were present abundantly in Glisson’ssheath and in the hepatic parenchyma of neonatal (3 and 7 days old) guinea pigs in a distributionsimilar to that in mature animals. This ontogenetic pattern suggests that NPY plays a certain role inthe developing liver. Microsc. Res. Tech. 39:365–371, 1997. r 1997 Wiley-Liss, Inc.

INTRODUCTIONNeuropeptide Y (NPY), a 36-amino-acid peptide origi-

nally isolated from the porcine brain (Tatemoto et al.1982), is richly present in both the central and periph-eral nervous systems (Adrian et al., 1983; Ekblad et al.,1984; Sundler et al., 1983; Wang et al., 1987). Immuno-histochemical studies have shown that NPY sometimescoexists with the catecholamines. In the peripheralnervous system, for example, NPY has been detectedmainly in perivascular noradrenergic nerves (Edvins-son et al., 1985; Lundberg, 1982a) although some NPYhas also been reported in nonadrenergic nerves of thegastrointestinal tract (Ekblad et al., 1984; Sundler etal., 1983).

NPY has been implicated in a variety of physiologicalevents in the peripheral nervous system, includingvasoconstriction (Edvinsson, 1984; Lundberg and Tate-moto, 1982); inhibition of smooth muscle contraction(Lundberg et al., 1982; Ohhashi and Jacobowitz, 1983),neuroendocrine regulation (Kalra and Crowley, 1984;Kerkerian et al., 1985), alteration of feeding behavior(Sahu, 1988), depression of heart rate (Macrae andReid, 1988), and inhibition of insulin release (Petters-son, 1987). In addition, NPY has been shown to be muchmore potent than noradrenaline in producing hepaticarterial vasoconstriction (Corder and Withrington,1988).

Recent immunohistochemical studies have furtherdemonstrated that NPY-containing nerves are presentin the liver of various animals including humans (Dinget al., 1991, 1994; Goehler and Sternini, 1991; Miya-zawa et al., 1988). However, the density of NPY-positivenerves differs among these animal species. In thehepatic parenchyma, for example, NPY fibers are abun-

dant in the monkey and human (Ding et al., 1994), butsparse in the rat and rabbit (Goehler and Sternini,1991). In addition to such species differences, possiblechanges in the density of NPY-positive nerve fibers mayoccur in the developing liver.

The present study summarizes the distribution ofNPY-positive fibers in the liver of different maturevertebrates and of developing mice and guinea pigs.

IMMUNOHISTOCHEMICAL PROCEDURESWe performed immunohistochemistry to study NPY

distribution in the liver by using the avidin-biotin-peroxidase (ABC) method (Ding et al., 1994). Liverspecimens were fixed in a mixture of 4% paraformalde-hyde and 0.2% picric acid buffered with 0.1 M sodiumphosphate, cryoprotected in 15% sucrose in 0.1 Mphosphate buffer, and cut into sections 20 µm thickusing a cryostat. The sections were put through thefollowing steps in a free-floating state: (1) incubationfor 2 days with NPY antibody (Peptide Inst. Inc., Japan;diluted 1:20,000) at 4°C, (2) incubation for 1 hour withbiotinylated anti-rabbit IgG (diluted 1:1000) at roomtemperature, (3) incubation for 1 hour with the ABCmethod (diluted 1:4,000), and (4) reaction for 5 minutesat room temperature with a mixture containing 0.02%3,38-diaminobenzidine 4 · HCl, 0.3% nickel ammoniumsulfate, and 0.0045% H2O2 in 0.05 M Tris-HCl buffer(pH 7.6).

*Correspondence to: Hiroshi Kimura, M.D., Ph.D., Department of Neuro-anatomy, Institute of Molecular Neurobiology, Shiga University of MedicalScience, Seta, Otsu, Shiga 520-21, Japan. E-mail: c51095@sakura.kudpc.kyoto-u.ac.jp.

Received 1 February 1995; accepted in revised form 9 November 1995

MICROSCOPY RESEARCH AND TECHNIQUE 39:365–371 (1997)

r 1997 WILEY-LISS, INC.

DISTRIBUTION OF NPY-POSITIVE NERVEFIBERS IN VARIOUS SPECIES

NPY-immunoreactive nerve fibers were found in theliver of every animal examined except the eel (Table 1).However, these positive fibers revealed a considerablespecies difference in both density and distributionpattern. In the carp, bullfrog (Fig. 1A), turtle (Fig. 1B),and chicken, positive fibers were few in number; whenpresent, they were localized only in Glisson’s sheath inareas surrounding blood vessels (Figs. 1A,B) or in thebile ducts. The density of positive fibers appeared to begenerally high in the mammalian species (Table 1). Inmice and rats (Fig. 1C), a moderate density of positivefibers was found in Glisson’s sheath, but no positivefibers were observed in the hepatic parenchyma includ-ing the wall of the central vein (Table 1). However, inmammals such as the guinea pig (Figs. 3E, 3G), dog(data not shown), monkey (Figs. 1D, 1E) and human(Fig. 1F), positive fibers were densely distributed notonly in Glisson’s sheath but also in the hepatic paren-chyma (Table 1). These positive fibers formed a networkin Glisson’s sheath to distribute themselves around thewalls of the interlobular portal vein, interlobular he-patic artery and hepatic bile duct (Figs. 1C, 1D). Fromthe network of positive fibers around blood vessels,ramified positive fibers were often seen extending intothe hepatic parenchyma. A few positive fibers weredetected in the wall of the central vein Only in the dog,monkey (Fig. 1E) and human (Table 1). In the hepaticparenchyma, the density of positive fibers was higher inthe peripheral than in the central lobular region. Thepositive fibers in the parenchyma, often thin and vari-cose, were situated adjacent to hepatocytes (Fig. 1F).

Both the density and distribution pattern of NPY-positive fibers varies considerably among species, assuggested by a study of three mammalian species(Goehler and Sternini, 1991). In general, NPY innerva-tion is dense in such higher mammals as the guinea pig,dog, monkey, and human but sparse in lower verte-brates such as the carp, bullfrog, turtle, and chicken. Inthe hepatic parenchyma, however, no positive fibers arerecognizable in these lower vertebrates or in somemammals such as mice and rats, whereas many posi-tive fibers are present in other mammals including the

human. These results suggest that the difference in thepattern of NPY fiber distribution among various speciesmay more or less reflect phylogenetic differences, al-though no functional significance is known.

At the immunoelectron microscopic level, some posi-tive terminals were occasionally located close to unla-beled nerve terminals (Fig. 2A, arrowhead), suggestinga putative role for NPY in the presynaptic regulation ofother neurotransmitters. It may be noteworthy thatNPY-positive nerve terminals are situated in closeapposition to hepatic vascular walls (data not shown)and hepatocytes (Fig. 2B). This finding implies thatboth hepatic endothelial cells and hepatocytes arefunctionally affected by neuron-mediated NPY releasefrom the terminals. In fact, it has been reported thatNPY potentiates noradrenaline-induced vasocontrac-tion in the rat portal vein (Dalhof et al., 1985) or evenproduces stronger vasocontraction than noradrenalinealone in the dog hepatic artery (Corder and Withring-ton, 1988).

ONTOGENETIC STUDY OF NPYINNERVATION

As shown in Table 2, NPY-positive nerve fibers in themouse liver were first detected in 19-day-old embryos(Fig. 3A). After birth, the density of positive fibersincreased with age to reach maximal level at 1 week(Fig. 3B) but later decreased and sustained adult levelsafter 2 weeks postnatal (Fig. 3C). The pattern of NPYfiber distribution also altered with age. Prenatally, afew positive fibers were observed in the vascular wallsof some but not all Glisson’s sheaths. After birth,positive fibers could be seen in every Glisson’s sheath ofmice older than 1 week.

NPY-positive afferent fibers are seen as early as thelate fetal period in the mouse, as suggested fromstudies on rat pancreatic tissue collected during latestages of embrogenesis (El-Salhy et al., 1987). Thepositive fibers in Glisson’s sheath show a transientincrease at 1 week after birth. Although the significanceof the transient expression remains unclear, NPY mayplay an important role in the developing liver.

Because our phylogenetic study has indicated thatNPY innervation in the hepatic parenchyma is ob-served mainly in higher mammals such as the guineapig, dog, monkey, and human, we chose the guinea pigfor the ontogenic study of hepatic NPY innervation. Ininfant (3 and 7 days old) guinea pigs, NPY-positivenerve fibers and bundles were abundant in the stromalcompartments, which were associated with the vascula-ture and billiary pathway (Fig. 3D, Table 3). Thepattern of distribution in infants is similar to that inadults (Fig. 3E). In addition, moderate supplies ofNPY-positive nerve fibers were also demonstrated inthe hepatic parenchyma of neonates (Fig. 3F). Thereappeared to be no significant difference in the densityand distribution of hepatic NPY innervation betweenneonates and adults (Fig. 3G).

ORIGIN OF NPY INNERVATION IN LIVERBecause in the liver no neuronal somata have been

observed to be positive for NPY in any animal exam-ined, NPY-positive nerve fibers appear to be derivedfrom extrinsic sources. As shown in Figures 4A and 4B,numerous NPY-positive nerve fibers were seen in the

TABLE 1. Presence of NPY-immunoreactive nerve fibers in the liver offive main vertebrate groups1

Animalspecies

Glisson’ssheath

Liverparenchyma

Centralvein wall

TeleostsEel 2 2 2Carp 1 2 2

AmphibianBullfrog 1 2 2

ReptileTurtle 1 2 2

BirdChicken 1 2 2

MammalsMouse 11 2 2Rat 11 2 2Guinea pig 111 11 2Dog 111 11 1Monkey 111 111 1Human 111 111 1

12, none; 1, few; 11, moderate number; 111, numerous.

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walls of the dog portal vein and hepatic artery. Thesenerve fibers, with their many varicosities, usually ranparallel to blood vessels. Therefore, some postgangli-onic NPY-positive nerve fibers may enter the liver viathe hepatic hilus with the portal vein and hepaticartery and then distribute themselves into the interlobu-lar connective tissue region. The precise origins of theseNPY-positive hepatosympathetic afferents have not beenreported. We agree with the opinion of Goehler andSternini (1991) who proposed that the hepatic NPY

afferents are likely to originate from prevertebral sym-pathetic ganglia because we have observed many NPY-positive cells in the rat celiac ganglion (Fig. 4C).Furthermore, NPY-positive fibers are not detectable inthe rat liver after sympathectomy (Fig. 4D). These datastrongly indicate that NPY may be contained in sympa-thetic afferents of the rat liver. This supposition isfurther supported by previous data (Goehler and Ster-nini, 1991; Ding et al., 1994) showing that the adminis-tration of a sympathetic neurotoxin 6-hydroxydopa-

Fig. 1. NPY-positive nerve fibers at the light microscopic level inthe liver of the bullfrog (A), turtle (B), rat (C), monkey (D, E), andhuman (F). A: Positive fibers in the wall of an interlobular blood vesselof the bullfrog. B: Positive fibers near an interlobular portal vein of theturtle. C: Positive fibers in the walls of an interlobular portal vein(PV), interlobular hepatic artery (HA), and interlobular bile duct (BD)

of the rat. D: Abundant positive fibers in Glisson’s sheath around thewall of the PV in the monkey. E: Positive fibers in the wall of thecentral vein (CV) of a monkey. F: Positive fibers with numerousvaricosities are distributed widely in the liver parenchyma of thehuman. Some fibers appear to contact the surface of the hepatocytes.Counterstained with neutral red. Bars, 50 µm.

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mine resulted in an almost complete elimination ofNPY-positive fibers in the rat liver. In contrast, thevagal denervation affected neither the density nor thedistribution pattern of the NPY-positive fibers (Ding etal., 1994).

POSSIBLE ROLES OF NPY-POSITIVENERVE FIBERS

In the liver of various vertebrate examined in thisstudy, NPY-containing nerve fibers are distributed seem-ingly in association with the hepatic vasculature, inter-lobular bile ducts, and hepatic parenchyma. Althoughphysiological roles of these NPY fibers in the threehepatic assemblies are uncertain, it is possible tospeculate their roles from the view of the structure–function relationship. The NPY fibers running alongthe blood vessels may contribute significantly to regula-tion of hepatic blood flow. In fact, NPY has been shownto potentiate noradrenaline-induced vasocontraction inthe rat portal vein (Dalhof et al., 1985) or even toproduce stronger vasocontraction than noradrenalinealone in the dog hepatic artery (Corder and Withring-ton, 1988). The presence of NPY-containing fibers dis-tributing around the interlobular bile ducts imply thatNPY is involved in the regulation of bile transport,

although stimulation of the greater splanchnic nerve isreported to have effects only on the common bile ductand gallbladder (Persson, 1973). The parenchymal NPYinnervation is particularly noticed in higher mamma-lian species such as the guinea pig, dog, monkey, andhuman, thereby providing a possible direct effect ofNPY on the control of secretory processes of hepaticcells, as reportedly seen in the thyroid gland (Grunditzet al., 1984).

The mammalian liver has been believed to be inner-vated by afferent nerves and efferent autonomic, bothsympathetic and parasympathetic, nerve fibers. Affer-ent nerves may participate in osmo- and chemorecep-tion. Efferent nerves are thought to be involved not onlyin the regulating of intrahepatic blood flow but also inthe function control of the liver cell (Lautt, 1980, 1983;Yamaguchi, 1992). Stimulation of either preganglionicsplanchnic or postganglionic hepatic nerve has beenshown to increase glucose output from the liver invarious animal models (Lautt, 1980, 1983). Becausesuch preganglionic stimulation increases the secretionof adrenaline and noradrenaline from the adrenalmedulla (Shimazu and Amakawa, 1975) and of gluca-gon from the endocrine pancreas (Frohman and Bernar-dis, 1971; Woods and Porte, 1974), the enhanced he-patic glucose output seems to be mediated by thesehormonal factors. Experimental evidence, however, hasindicated that, in adrenalectomized mammals, a simi-lar hyperglycemic response does occur following directstimulation of bilateral splanchnic nerves (Edwards,1972). In the perfused rat liver, moreover, electricalstimulation of the nerve bundles near the hepaticartery and portal vein results in an increase of glucoseoutput and a decrease of portal blood flow (Hartmann etal., 1982). These data suggest that glycogen breakdownis regulated directly by nerves rather than indirectly byhemodynamic changes or noradrenaline overflow.

In agreement with a previous study (Goehler andSternini, 1991), the present study supports the ideathat NPY may exist in sympathetic efferents to theliver. Therefore, NPY contained in such nerves prob-ably cooperates with noradrenaline in regulating the

TABLE 2. NPY-immunoreactive nerve fibers in the mouse liverduring development1

AgeNo. of

specimensDensity of

NPY-positive fibers

15th day of gestation 8 217th day of gestation 8 219th day of gestation 8 11st day postnatal 6 13rd day postnatal 6 111st week postnatal 4 1112nd week postnatal 4 113rd week postnatal 4 11

12, none; 1, few; 11, moderate number; 111, numerous.

TABLE 3. NPY-immunoreactive nerve fibers in the guinea pig liverduring development1

Age Glisson’s sheath Liver parenchyma

3rd day postnatal 111 111st week postnatal 111 11Adult 111 11/111

111, moderate number; 111, numerous.

Fig. 2. NPY immunoelectron micrographs of Glisson’s sheath (A)and liver parenchyma (B) of the human. Bars, 1 µm. A: Positiveterminals with numerous immunoreactive vesicles in Glisson’s sheath.Arrowhead indicates such a terminal in close contact with an unla-beled nerve terminal (star). B: A positive terminal located in adepression between two neighboring liver cells (LC). The two gaps onboth sides of the depression are collagen fibers.

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Fig. 3. NPY-positive nerve fibers at the light microscopic levelduring liver development in the mouse (A–C) and guinea pig (D–G). A:Positive fibers in Glisson’s sheath of a 19-day-old mouse embryo. B: Adense network of positive fibers in Glisson’s sheath of 7-day-old mouse.C: Decreased density of positive fibers in Glisson’s sheath of an adult

mouse. D,E: Positive fibers in Glisson’s sheath of 3-day-old guinea pig(D) and adult guinea pig (E). F,G: Positive fibers with numerousvaricosities are moderately distributed in the liver parenchyma of3-day-old guinea pig (F) and an adult guinea pig (G). Counterstainedwith neutral red. Bar, 50 µm.

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hepatic blood flow and some other function. Indeed,corelease of NPY with noradrenaline has been observedduring sympathetic nerve stimulation (Lundberd andHokfelt, 1986).

Recently, it has been suggested that the liver is majorsource of increased circulating NPY induced by sympa-thetic nerve stimulation (Taborsky et al., 1994). If so,the increased circulating NPY may affect various physi-ological states of the body, including blood pressure(Pernow et al., 1989), neuroendocrine actions (Kalraand Crowley, 1984; Kerkerian et al., 1985), feedingbehavior (Sahu, 1988), and insulin secretion (Petters-son et al., 1987).

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