Distribution of neuropeptide FF-like immunoreactivestructures in the lamprey central nervous systemand its relation to catecholaminergic neuronal structures

Manuel A. Pombal a,*, Jesus M. Lopez b, Marıa C. de Arriba a, Manuel Megıas a,Agustın Gonzalez b

aNeurolam Group, Department of Functional Biology and Health Sciences, Faculty of Biology, University of Vigo, 36310 Vigo, SpainbDepartment of Cell Biology, Faculty of Biology, University Complutense, 28040 Madrid, Spain

p e p t i d e s 2 7 ( 2 0 0 6 ) 1 0 5 4 – 1 0 7 2

a r t i c l e i n f o

Article history:

Received 1 April 2005

Accepted 22 June 2005

Published on line 17 February 2006

Keywords:

FMRFamide-related peptides

Neuropeptides

Immunohistochemistry

Hypothalamus

Agnathan

Evolution

a b s t r a c t

The neuropeptide FF (NPFF) is an octapeptide of the RFamide-related peptides (FaRPs) that

was primarily isolated from the bovine brain. Its distribution in the CNS has been reported in

several mammalian species, as well as in some amphibians. Therefore, in order to gain

insight in the evolution on the expression pattern of this neuropeptide in vertebrates, we

carried out an immunohistochemical study in the sea lamprey, Petromyzon marinus. The

distribution of NPFF-like-immunoreactive (NPFF-ir) structures in the lamprey brain is, in

general, comparable to that previously described in other vertebrate species. In lamprey,

most of the NPFF-ir cells were found in the hypothalamus, particularly in two large

populations, the bed nucleus of the tract of the postoptic commissure and the tuberomam-

millary area. Numerous NPFF-ir cells were also observed in the rostral rhombencephalon,

including a population in the dorsal isthmic gray and the reticular formation. Additional

labeled neurons were found inside the preoptic region, the parapineal vesicle, the periven-

tricular mesencephalic tegmentum, the descending trigeminal tract, the nucleus of the

solitary tract, as well as in the gray matter of the spinal cord. The NPFF-ir fibers were widely

distributed in the brain and the spinal cord, being, in general, more concentrated throughout

the basal plate. The presence of NPFF-ir fibers in the lamprey neurohypophysis suggests that

the involvement of NPFF-like substances in the hypothalamo-hypophyseal system had

emerged early during evolution.

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1. Introduction

The octapeptide neuropeptide FF (NPFF, FLFQPQRF-amide)

has been referred to as mammalian FMRF-NH2-like peptide,

F-8-F-amide, or morphine modulating neuropeptide. The

NPFF family consists of several different peptides: NPFF

and neuropeptide AF (NPAF), both originally isolated from

bovine brain by using antisera directed against the molluscan

* Corresponding author. Tel.: +34 986 812390; fax: +34 986 812556.E-mail address: pombal@uvigo.es (M.A. Pombal).

0196-9781/$ – see front matter # 2006 Elsevier Inc. All rights reserveddoi:10.1016/j.peptides.2005.06.033

tetrapeptide FMRF-NH2 [88]; neuropeptide SF (NPSF), primarily

identified from the rat brain and spinal cord [89]; as well as

different longer peptides such as NPA-NPFF wich is the most

abundant in the rat spinal cord [9]; and SQA-NPFF, identified in

human neuroblastoma transfected with human proNPFF

transcript, and its homologue in mouse, SPA-NPFF [10]. Two

precursors (proNPFFA and proNPFFB) encoding peptides con-

taining the PQRF-amide sequence have been recently cloned

.

p e p t i d e s 2 7 ( 2 0 0 6 ) 1 0 5 4 – 1 0 7 2 1055

in mammals [58,81]. In a human cell line (neuroblastoma

SH-SY5Y), NPFF, NPAF, and SQA-NPFF are generated from

proNPFFA, whereas, NPFF, NPSF, and SPA-NPFF are identified

in the spinal cord of mice [10].

NPFF-like immunoreactivity (NPFF-ir) has been demon-

strated in the CNS of several mammalian species, including

rats ([33,34,42,45]; reviewed in [55,56]) and bovine brain [46],

as well as the spinal cords of mice, rats, guinea pigs and

humans [1,3,9,31,36,44]. Although NPFF has been detected in

the human plasma [72], its immunoreactivity seems to be

largely confined to the CNS, thus differing from a large

number of brain-gut peptides, which are found both in the

central and peripheral nervous system. In mammals, cell

bodies displaying NPFF-ir are generally restricted to a few

locations in the CNS: the central hypothalamus, supraoptic

and paraventricular nucleus, the nucleus of the solitary tract,

the spinal nucleus of the trigeminal tract, and different zones

of the dorsal horn in the spinal cord (see [55]). However,

numerous NPFF-ir fibers are widely distributed throughout

the mammalian CNS, with the highest innervations in the

posterior pituitary and dorsal horn of the spinal cord,

followed by the hypothalamus and the hindbrain [45,55].

Recently, a comparative study on the NPFF-ir structures in

the CNS of both anuran and urodele amphibians have also

been reported [18], and it represents the only report in

anamniote vertebrates. The presence of numerous NPFF-ir

fibers in the hypothalamo-neurohypophyseal system has

been observed in some of these studies [6,18,49].

In mammals, NPFF has been implicated in pain modulation

[1,23,29,88], opiate function [50,71,73], behavior [30], cardio-

vascular regulation [66], and neuroendocrine function

[2,47,48,49]; reviewed by Panula et al. [55]. The pharmacolo-

gical effects of NPFF and its analogs result from their

interactions with two G-protein-coupled receptors, NPFF1

and NPFF2. These receptors have been recently cloned and

characterized in rats and humans [8,20,25,38]. Available

information about the effects of NPFF on central neurotrans-

mitter systems is limited. According to pharmacological

experiments, NPFF has hyperalgesic effects [73,88] and it

elevates arterial blood pressure when injected intravenously

in rats [66]. In addition, both catecholamine-dependent and -

independent mechanisms appear to be involved in the

hypertensive effect of NPFF [66]. Tyrosine hydroxylase (TH)

is the rate-limiting enzyme in catecholamine biosynthesis,

and its immunohistochemical presence has been largely used

to identify catecholaminergic neurons (e.g., [60]). The presence

of NPFF-ir cells in many areas where TH immunoreactive (TH-

ir) neurons were previously found (see [59,60,62,84]), together

with the colocalization of these two substances in a

subpopulation of neurons in the nucleus of the solitary tract

of rats [32,42] promted us to check their presence in the same

cells in the lamprey’s CNS.

Among RFamide-related neuropeptides, antisera to the

molluscan cardio-excitatory tetrapeptide, FMRFamide, have

been used to identify FMRFamide-like immunoreactive mate-

rial in the CNS of many different invertebrate and vertebrate

species [5,24,26], including agnathans [12,15,19,28,54,85] and

many other fishes [13,16,61,77,78,87]. However, the presence

of NPFF has only been reported for some amphibian and

mammalian species [18,33,34,42,45,46,55,56].

The aim of this work was to study the distribution of NPFF-

ir structures in the brain of the adult sea lamprey, Petromyzon

marinus. Lampreys, together with hagfishes, are the only two

living representatives of the oldest lineage of vertebrates

(agnathans, jawless vertebrates). Interest in these groups of

fish is stimulated by their ancient lineage and the potential for

gaining insight into the evolution and distribution of neuro-

peptides, such as NPFF, in vertebrates.

2. Materials and methods

2.1. Animals

A total of 10 sexually mature adult sea lampreys (P. marinus;

seven males and three females) were used in the present

study. The animals were captured in the Mino River (north-

west part of Spain) during their upstream migration for

breeding, and obtained from a local supplier (Lampreas y

Angulas del Mino: Benıtez Fernandez, S.L.). The original

research reported here followed the guidelines on animal

care established by the Spanish Royal Decree 223/1988.

The animals were carried to the laboratory and processed

upon arrival. They were first deeply anesthetized in a 0.1%

solution of tricaine methanesulfonate (MS222; Sigma–Aldrich

Co., St. Louis, MO). Four animals were then perfused

transcardially with lamprey Ringer’s solution followed by

300 ml of 4% paraformaldehyde in 0.1 M phosphate buffer (PB;

pH 7.4), whereas, the other 6 animals were killed by

decapitation.

2.2. NPFF immunohistochemistry

The brain and rostral spinal cord was quickly dissected out

and fixed for 3–24 h at 4 8C in the same fixative solution. The

fixed tissues were then immersed in a solution of 30%

sucrose in PB at 4 8C until they sank, embedded in a solution

of 20% gelatin with 30% sucrose in PB, and immersed in a 4%

formaldehyde solution at 4 8C for 5 h. The gelatin blocks were

cut on a freezing microtome at 40 mm in the frontal or

sagittal plane and collected in PB. The tissue slices were then

rinsed twice in PB, treated with 1% H2O2 in PB for 15 min to

reduce endogenous peroxidase activity, and rinsed again

three times (10 min each) in PB. Sections were then

processed for immunohistochemistry by the peroxidase

anti-peroxidase (PAP) method [70]. Briefly, this included a

serial incubation of the sections in (1) a rabbit anti-NPFF

serum (kindly donated by Dr. H.-Y.T. Yang, NIH, Bethesda,

MD, USA) diluted 1:1000 in PB containing 2.5% Triton X-100,

for 48 h at 4 8C; (2) 0.1 M PB (3� 10 min); (3) swine anti-rabbit

serum (Nordic, Tilburg, The Netherlands), diluted 1:50 for

1 h; (4) 0.1 M PB (3� 10 min); (5) rabbit PAP complex

(Dakopatts, Denmark), diluted 1:500 for 90 min; (6) 0.1 M PB

(3� 10 min); (7) 0.5 mg/ml 3,30-diaminobenzidine (DAB;

Sigma, St. Louis) intensified with nickel, in 0.01% H2O2 in

distilled H2O; and (8) 0.05 M Tris–HCl buffer (TB, pH 7.6; 3�10 min). The secondary antiserum and the PAP complex were

diluted in PB containing 2.5% Triton X-100. Finally, the

sections were mounted (mounting medium, 0.25% gelatin in

TB) and, after drying overnight, added coverslips.

p e p t i d e s 2 7 ( 2 0 0 6 ) 1 0 5 4 – 1 0 7 21056

Fig. 1 – (A–O) Composite plate illustrating a set of representative transverse sections through the brain and rostralmost

spinal cord of Petromyzon marinus. The rostrocaudal level of each section is indicated in the schematic lateral view of the

brain as shown in (P). Photomicrographs of the left half of (A–O) show the overall distribution of NPFF-ir cells and fibers,

whereas, the specific names for neuronal groups or brain areas (drawn with dashed lines), major fiber tracts (drawn with

dotted lines), or individual large neurons (M3) are represented in the accompanied explanatory drawings (right part).

p e p t i d e s 2 7 ( 2 0 0 6 ) 1 0 5 4 – 1 0 7 2 1057

2.3. Double labeling for NPFF and TH

In four cases, a procedure based on immunohistofluores-

cence was used in a series of alternate sections as follows: (1)

incubation in a cocktail of mouse anti-TH (diluted 1:1000;

Incstar, Stillwater, MN, USA) and rabbit anti-NPFF (diluted

1:1000) in PB containing 2.5% Triton X-100, for 48 h at 4 8C; (2)

second incubation in a mixture of Alexa Fluor 594-conjugated

goat anti-rabbit (red fluorescence, diluted 1:300; Molecular

Probes, Eugene, OR, USA) and Alexa Fluor 488-conjugated goat

anti-mouse (green fluorescence, diluted 1:300; Molecular

Probes) for 90 min at room temperature; (3) after rinsing

three times in PB, the sections were mounted on glass slides

and coverslipped with Vectashield (Vector, Burlingame, CA,

USA); and (4) finally, the staining was studied and photo-

graphed with epifluorescent illumination by using the

appropriate filter combinations.

2.4. Evaluation and presentation of the results

The rabbit NPFF antiserum used in this study was previously

characterized and used in mammalian neuroanatomical

studies [27,46]. In addition, different sets of controls were

carried out in the amphibian brain [18] by preabsorption of the

antibody with bovine NPFF or a range of neuropeptides

structurally related and unrelated to the C-terminal part of

NPFF: FMRFamide, neuropeptide Y, adrenomedullin and

somatostatin, to test the specificity of the immunostaining.

The same antibody was used to detect the NPFF-ir structures

in the lamprey CNS following the immunohistochemical

procedures described in Sections 2.2 and 2.3.

The distribution of NPFF-like immunoreactive (NPFF-ir) cell

bodies and fibers in the brain of the adult sea lamprey is shown

Abbreviations: AB, anterobasal nucleus; aot, anterior octavomotor

cerebellar commissure; cc, central canal of the spinal cord; ch, o

region (gray matter) of the spinal cord; cs, central stratum (optic

dorsal column fibers; dh, dorsal horn of the spinal cord; dig, do

dmc, dorsomedial column of the spinal cord; dn, dorsal nucleus

tegmentum; dV, descending trigeminal tract; f, fasciculus retrofl

commissure; igl, internal granular layer of the olfactory bulb; II

trochlear nucleus; IX, glossopharyngeal motor nucleus; lc, latera

part of the lateral pallium; lpv, ventral part of the lateral pallium

mammillary region sensu lato; mlf, medial longitudinal fascicul

pallium; MPO, medial preoptic nucleus; mra, mesencephalic reti

mt, mesencephalic tegmentum; NH, neurohypophysis; nLLA, an

longitudinal fasciculus; NSM, nucleus of the stria medullaris; NT

of the tract of the postoptic commissure; nts, nucleus of the solit

area; on, optic nerve; os, optic stratum (optic tectum); ot, optic tec

paracommissural preoptic nucleus; PMg, magnocellular preopti

rhombencephalic reticular nucleus; ps, periventricular stratum

nucleus; PV, paraventricular nucleus; PVO, hypothalamic perive

mesencephalic recess; RM, retromammillary area; rm, mammil

rpro, preoptic recess; S, striatum; SCO, subcommissural organ;

intermedius ventralis; slh, sulcus limitans of His; SO, spino-occ

dorsal thalamus; TM, tuberomammillary nucleus; TN, tuberal nu

motor nucleus; vc, ventral column of the spinal cord; vd, dience

motor nucleus; vla, anterior lateral telencephalic ventricle; vlp, p

ventricle; vn, ventral nucleus of the octavolateral area; vr, rhom

vagal motor nucleus (rostral part). Scale bar represents 1 mm.

in Fig. 1. The drawings accompanying the pictures to show the

location of different areas, nuclei and major fiber tracts were

made by means of a camera lucida. Additional series of lamprey

stained brains from our collection were available for topo-

graphical purposes. In the present study, we followed the

nomenclature of Pombal and Puelles [65] for the forebrain, and

that of Pombal et al. [62,64] for the brainstem and spinal cord.

3. Results

The NPFF immunohistochemical procedure consistently

stained different populations of neurons in the lamprey

brain and spinal cord, and numerous NPFF-ir fibers were

widely distributed throughout the CNS. Most of the NPFF-ir

cells were observed in the hypothalamus, followed by the

isthmic region. The distribution and morphology of labeled

cells and fibers did not appear to vary between males and

females examined. Low power transverse pictures showing

the overall distribution of NPFF-ir cells and fibers in the

lamprey brain and spinal cord, as well as corresponding

detailed schematic drawings are shown in Fig. 1.

3.1. Distribution of NPFF-ir perikarya

3.1.1. Telencephalon

No NPFF-ir cell bodies were found in the olfactory bulb. The

most rostrally located group of NPFF-ir neurons was con-

stituted by scattered cells in the paraterminal preoptic nucleus

(Figs. 1A and 2A). The cells were spindle-shaped and

moderately immunostained and distributed throughout the

commissure of the lamina terminalis located in front of the

preoptic recess. More caudally, some weakly stained cells

tract; arn, anterior rhombencephalic reticular nucleus; cbc,

ptic chiasma; cpi, postinfundibular commissure; cr, cell

tectum); ctp, commissure of the tuberculum posterior; dcf,

rsal isthmal gray; dm, dorsomedial telencephalic neuropil;

of the octavolateral area; ds, dorsal sac; dt, diencephalic

exus; go, glomeruli olfactorii; HB, habenula; ibc, interbulbar

I, oculomotor nucleus; ip, interpeduncular nucleus; IV,

l column of the spinal cord; lp, lateral pallium; lpd, dorsal

; M3, third Muller cell; M5, nucleus M5 of Schober; MAM,

us; mn, medial nucleus of the octavolateral area; M, medial

cular area; mrn, medial rhombencephalic reticular nucleus;

terior lateral line nerve; nmlf, nucleus of the medial

P, nucleus of the tuberculum posterior; nTPOC, bed nucleus

ary tract; nV–nVIII, cranial nerves V–VIII; ola, octavolateral

tum; pc, posterior commissure; pch, choroidal plexus; PCP,

c nucleus; poc, postoptic commissure; prn, posterior

(optic tectum); PT, pretectum; PTP, paraterminal preoptic

ntricular organ; ri, infundibular recess; rlm, lateral

lary recess; rnp, neuroporic recess; rpo, postoptic recess;

SE, septum; SHL, subhippocampal lobe; siv, sulcus

ipital nucleus; ss, superficial stratum (optic tectum); TD,

cleus; to, optic tract; ts, torus semicircularis; V, trigeminal

phalic ventricle; VI, abducens motor nucleus; VII, facial

osterior lateral telencephalic ventricle; vm, mesencephalic

bencephalic ventricle; vt, impar telencephalic ventricle; Xr,

p e p t i d e s 2 7 ( 2 0 0 6 ) 1 0 5 4 – 1 0 7 21058

Fig. 1. (Continued).

were found in the compact band of closely packed cells that

constitute the magnocellular preoptic nucleus (Figs. 1B and

2B). In transverse sections, these cells were distributed

dorsally to the optic chiasm and were interspersed among

negative cell bodies. The NPFF-ir cells gave rise to two

processes: one apical process that coursed medially and

contacted the cerebrospinal fluid (CSF) in the preoptic recess;

and a basal process that coursed in the lateral neuropil

(Fig. 2B). A few NPFF-ir cells were also observed in the

paracommissural preoptic nucleus (Figs. 1C and 2C). These

were small piriform cells located just dorsal to the dorsal

postoptic commissure and were of CSF-contacting type

contacting with the impar telencephalic ventricle in the

medial part of the brain.

p e p t i d e s 2 7 ( 2 0 0 6 ) 1 0 5 4 – 1 0 7 2 1059

Fig. 2 – Photomicrographs showing NPFF-ir cells and fibers in transverse (A–C, F) and sagittal (D, E, G, H) sections through the

forebrain (A–G) and midbrain (H) of Petromyzon marinus. (A) Positive cells in the paraterminal preoptic nucleus (PTP) and

periventricular immunoreactive plexus in the septum (SE): rnp, neuroporic recess; (B) small weakly labeled cells (arrows) in

the magnocellular preoptic nucleus: rpro, preoptic recess; vt, impar telencephalic ventricle. (C) Many strong-labeled CSF-

contacting cells in the bed nucleus of the tract of the postoptic commissure (arrow) and few in the paracommissural

preoptic nucleus (arrowhead). (D) Numerous labeled cells in the bed nucleus of the tract of the postoptic commissure

(nTPOC), as well as in its rostral continuation, the anterobasal nucleus (AB): AH, adenohypophysis; ch, optic chiasma;

p e p t i d e s 2 7 ( 2 0 0 6 ) 1 0 5 4 – 1 0 7 21060

3.1.2. HypothalamusThe most conspicuous hypothalamic population of NPFF-ir

neurons was found in the anterobasal nucleus (Fig. 1B) and the

bed nucleus of the tract of the postoptic commissure (Fig. 1C

and D). This NPFF-ir population consists of numerous and

highly immunoreactive neurons located ventral and caudal to

the optic chiasm (Fig. 2C–E). These cells were round, piriform

or polygonal in shape, and possessed one thick and short

apical dendrite that reached the ventricular surface (Fig. 2C).

Immunopositive perikarya were intermingled among NPFF-

negative cells and arranged in three or four rows parallel to the

infundibular ventricular wall. The cell body of those NPFF-ir

cells located in the ependymal layer was small; whereas, the

most lateral ones showed larger somata (see the slightly

oblique parasagittal section shown in Fig. 3E). A few NPFF-ir

cells appeared displaced more caudally, thus entering the

limits of the hypothalamic periventricular organ. Numerous

processes originated from these cells coursed in several

directions, but mostly in the rostrocaudal plane innervating

the basal plate.

A second hypothalamic NPFF-ir group was detected in the

tuberomammillary area (Fig. 1E). The labeled cells occupied a

position ventral and slightly caudal to the bed nucleus of the

tract of the postoptic commissure. The cells were weakly

labeled and arranged parallel to the ventricle (Figs. 1E and 2F).

Moreover, some of the NPFF-ir cells contacted the CSF by

means of a club-shaped process (Fig. 2F).

3.1.3. DiencephalonWithin the diencephalon, only few NPFF-ir cells were observed

in the parapineal organ (Fig. 2G). These cells were weakly

labeled and had elongated bipolar cell bodies located in the

ventral wall of the parapineal vesicle. The pale cells gave rise

to more intensely stained processes with numerous and thick

varicosities that appears to innervate the neuropil of the

parapineal ganglion (Fig. 2G). Some labeled fibers were also

seen in the tela choroidea between the parapineal and the

habenula. The thalamus and the pretectum, as well as their

corresponding basal plate portions were devoid of NPFF-ir

cells in P. marinus (see Fig. 1E and F).

3.1.4. MesencephalonWithin the mesencephalon, one group of moderate NPFF-ir

cells was present in the subperiventricular tegmentum

(Fig. 1G). The neurons were small and possessed round cell

bodies with few and short processes (Fig. 2H). The NPFF-ir cells

were distributed dorsally to the dorsal rectus motor sub-

nucleus of the oculomotor nucleus, and thus can be

considered as belonging to the M5 nucleus of Schober, but

representing only a portion (ventral portion) of this retinopetal

nucleus.

Fig. 2. (Continued)

cpi, postinfundibular commissure; f, fasciculus retroflexus; NH,

bed nucleus of the tract of the postoptic commissure in a slightly

(thin arrow) and lateral (thick arrow) labeled cells. (F) Numerou

nucleus: ri, infundibular recess. (G) Weakly labeled cells (arrow

thick varicosities in the parapineal ganglion (G). (H) Immunorea

just dorsal to the third Muller cell (M3): vm, mesencephalic vent

200 mm (E, G, H).

3.1.5. RhombencephalonIn the dorsal part of the isthmic region, a group of numerous

NPFF-ir cells occupied a position from lateral to the trochlear

motor nucleus to the rostral portion of the trigeminal sensory

nucleus (Fig. 1H). The cells were scattered and weakly labeled

forming an arc throughout the isthmal gray, ventral to the so-

called cerebellar plate (Figs. 1H, 3A and B). The NPFF-ir cells

were multipolar, with fine, branching processes extending in

all directions, but forming a prominent plexus at the

periventricular side (Figs. 1H, 3A and B). The axons of these

cells appeared to fasciculate in the lateral part of the cell

group, forming a conspicuous and intense labeled fiber tract

that coursed ventromedially towards the midline (Fig. 1H and

I). From there, the axons could not be further followed because

of the dense network of labeled fibers and terminals within

which they were embedded.

More ventrally, some moderate to strong NPFF-ir cells were

located periventricularly, within the limits of the anterior

nucleus of the rhombencephalic reticular formation (Figs. 1H,

I, 3A and C, see also Fig. 4F). The cells were mostly located in

the caudal part of the nucleus. Most labeled reticular cells

were small, round or bipolar in shape, and grouped just below

the ependymal surface of the fourth ventricle (Fig. 3C).

Some NPFF-ir cell somata were primarily localized within

the limits of the descending trigeminal tract (Figs. 1J, 3D and E),

thus corresponding to the descending trigeminal tract

nucleus. The cells were only observed in the rostral one third

of this nucleus. The majority of these cells were small

multipolar neurons with short processes directed in several

directions (Fig. 3D and E).

In the caudal part of the rhombencephalic alar plate, some

strongly labeled neurons were found within the limits of the

nucleus of the solitary tract (Fig. 1M). Most of these cells were

grouped and laterally displaced, and located dorsal to the

descending trigeminal tract (Fig. 3F). The cells formed a

longitudinal band that can be clearly delineated in sagittal

sections (Fig. 3G) ending close to the level of the obex.

3.1.6. Spinal cordSome NPFF-ir cells were present in the dorsal aspect of the

spinal gray matter (Figs. 1O and 3H). These cells were mostly

monopolar, with a thick process that ramified dorsally and

originated the axon (Fig. 3H). Some bipolar cells were also

observed found on both sides of the midline, just ventral to the

lateral part of the dorsomedial column.

3.2. Distribution of NPFF-ir fibers

The NPFF-ir fibers were widely distributed in the adult sea

lamprey. Most of these fibers coursed longitudinally along

the laterobasal part of the brain, extending from the

neurohypophysis; rm, mammillary recess. (E) Detail of the

parasagittal section showing different size for the medial

s CSF-contacting cells (arrows) in the tuberomammillary

s) in the parapineal vesicle (V) and labeled fibers bearing

ctive cells in the mesencephalic periventricular nucleus,

ricle. Scale bar represents 100 mm (A–C, F), 500 mm (D), and

p e p t i d e s 2 7 ( 2 0 0 6 ) 1 0 5 4 – 1 0 7 2 1061

Fig. 3 – Photomicrographs showing NPFF-ir cells and fibers in transverse (A, C, D, F) and sagittal (B, E, G, H) sections through

the hindbrain of Petromyzon marinus. (A and B) Labeled cells in the dorsal isthmal gray nucleus: aot, anterior octavomotor

tract; arn, anterior rhombencephalic reticular nucleus; cbc, cerebellar commissure; vr, rhombencephalic ventricle. (C)

Immunoreactive cells (arrows) in the posterior part of the anterior rhombencephalic reticular nucleus, at the level of the

second isthmic Muller cell (I2). (D and E) Small labeled cells in the nucleus of the descending trigeminal tract: dV,

descending trigeminal tract. (F and G) Positive cells (arrows) in the nucleus of the solitary tract: dV, descending trigeminal

tract; slh, sulcus limitans of His; vn, ventral nucleus of the octavolateral area; Xr, vagal motor nucleus (rostral part). (H) Two

labeled cells and numerous fibers in the spinal cord. Scale bar represents 100 mm (A–G) and 25 mm (H).

p e p t i d e s 2 7 ( 2 0 0 6 ) 1 0 5 4 – 1 0 7 21062

Fig. 4 – Photomicrographs showing NPFF-ir fibers in transverse (A, C, D, G, H) and saggital (B, E, F) sections through the brain

and rostral spinal cord of the lamprey Petromyzon marinus. (A) Numerous labeled fibers in the neuropil of the striatum (S):

MPO, medial preoptic nucleus; vt, impar telencephalic ventricle. (B) Positive fibers in the ventral hypothalamus, including

the neurohypophysis (NH) and the postinfundibular commissure (cpi): rm, mammillary recess. (C) Detail of the

p e p t i d e s 2 7 ( 2 0 0 6 ) 1 0 5 4 – 1 0 7 2 1063

telencephalon up to the level of the spinal cord (Fig. 1). In

general, fibers showing NPFF immunoreactivity contained

bead-like varicosities along their trajectory, suggesting that

they form en passant synaptic contacts. The majority of

these fibers did not form discrete bundles, but were scattered

diffusely throughout diverse regions of the brain, with

particular accumulations in some nuclei as described herein.

3.2.1. TelencephalonIn the olfactory bulbs of the adult lampreys, only some

scattered NPFF-ir fibers were seen throughout of the internal

granular layer (Fig. 1A and B). These fibers did not enter in the

olfactory glomeruli, but a few were seen inside the olfactory

nerve. Some NPFF-ir fibers were also present in the pallium,

being more abundant in the ventral part of the lateral pallium

and the subhipoccampal lobe (Fig. 1B–D). In the subpallium,

labeled fibers were more numerous (Figs. 1A–C, 2A and 4A). In

the septal region, the labeled fibers accumulated in the vicinity

of the subventricular surface (Figs. 1A and 2A), whereas, in the

striatum region they were homogeneously distributed

throughout the periventricular neuropil (Figs. 1B, C and 4A).

3.2.2. HypothalamusA conspicuous fiber innervation was found throughout the

lamprey hypothalamus (Figs. 1B–F, 2B–F, 4B and C). These

fibers seem to be originated from the numerous NPFF-ir cells

located in this region. Most of these fibers coursed laterally,

and then turn either ventrally to enter the neurohypophysis

and the postinfundibular commissure, or caudally to inner-

vate the brainstem and the spinal cord. The neurohypophysis

was richly innervated from rostral to caudal with the

immunoreactive fibers showing numerous thick varicosities

(Figs. 2D and 4B). In sagittal sections, part of the labeled fibers

that innervate the neurohypophysis could be followed to their

place of origin in the bed nucleus of the tract of the postoptic

commissure. The postinfundibular commissure was also richly

innervated with the largest accumulation of labeled fibers in

its superficial half portion (Figs. 1F, 2D, 4B and C). Other fibers

coursed rostrally to innervate the preoptic area as well as the

subpallial and pallial regions, and the olfactory bulbs. Finally,

some fibers coursed dorsally to innervate partially the dorsal

diencephalon and mesencephalon (see Fig. 1D–G). These

fibers were relatively thick in the vicinity of the cell body

and became thinner along their trajectory. In addition, some

labeled fibers were also detected in the optic nerves (Fig. 1B).

3.2.3. DiencephalonIn the diencephalon, the alar plate of both ventral and dorsal

thalamus were poorly innervated, however some NPFF-ir

postinfundibular commissure (cpi): rm, mammillary recess. (D)

ventricle. (E) Distribution of labeled fibers in the superficial (ss),

optic tectum: vm, mesencephalic ventricle. (F) Labeled fibers in

with different intensities of immunoreaction in the interpedunc

posterior part of the anterior rhombencephalic reticular nucleus

ventricle. (G) NPFF-ir fibers concentrate in the periventricular ne

motoneurons of the trigeminal motor nucleus (V): mrn, medial

His; vr, rhombencephalic ventricle. (H) Distribution of labeled fi

columns, as well as in the dorsal horn (dh) and the cell region (c

100 mm (A–E, G, H) and 500 mm (F).

fibers accumulated in the habenula and its vicinity (Figs. 1E

and 4D), as well as in the ganglion of the parapineal organ

(Fig. 2G). In this ganglion, numerous labeled fibers with thick

varicosities were evenly distributed (Fig. 2G). The pretectal alar

plate was homogeneously innervated by NPFF-ir fibers, with

the exception of the optic tract (that is almost devoid of labeled

fibers throughout its rostrocaudal extent), and the subcom-

missural organ, where only few fibers could be seen (Fig. 1F).

Ventrally, some fibers crossed in the commissure of the

tuberculum posterior and the subpretectal commissure,

whereas, dorsally the fibers crossed in the posterior commis-

sure (Fig. 1F).

3.2.4. MesencephalonA moderate number of NPFF-ir fibers were also present in the

mesencephalic tegmentum as well as in the dorsal optic

tectum (Fig. 1G). In the tegmentum, the labeled fibers were

evenly distributed in the medial and lateral portions, while the

subventricular region where most cell bodies were located was

poorly innervated (Fig. 1G). Some fibers were also seen inside

the oculomotor nucleus intermingled with the motoneurons

(Figs. 1G and 2H). In the optic tectum, the NPFF-ir fibers

showed a precise fiber arrangement that is conserved

throughout its rostrocaudal extent. The labeled fibers were

more abundant in the periventricular portion, including the

periventricular and central strata, than in the superficial

portion, including the optic and superficial strata (Figs. 1G and

4E). However, the dorsomedial border of the optic tectum

displayed the largest accumulation of immunoreactive fibers

(Fig. 1G); some of these fibers crossed the midline at the

caudally located posttectal commissure.

3.2.5. RhombencephalonThe isthmic region, together with the hypothalamus, showed

the highest concentration of NPFF-ir fibers in the lamprey

brain. One of the major accumulations of immunoreactive

fibers within the isthmic region occurred in the dorsal part,

just at the level of the labeled cells (Figs. 1H, I, 3A and B). A

particular dense network of thin fibers was present in the

ventromedial neuropil of the interpeduncular nucleus

(Figs. 1G–I, 3C and 4F). Rostrocaudally, the interpeduncular

nucleus spread from the oculomotor nucleus to the level of the

first half of the trigeminal motor nucleus. In our material,

different densities for NPFF-ir fibers were detected in

consecutive rostrocaudal regions of the interpeduncular

neuropil, with the highest concentration at both sides of the

midline (see Figs. 1G–I and 3C). More laterally, several portions

particularly well delineated were undoubtedly distinguishable

in sagittal sections (Fig. 4F). It includes two parts with very few

Labeled fibers in the habenula (HB): vd, diencephalic

optic (os), central (cs), and periventricular (ps) strata of the

the caudal mesencephalon and rostral rhombencephalon,

ular neuropil (ip). Some labeled cells are also present in the

(arn): vm, mesencephalic ventricle; vr, rhombencephalic

uropil, whereas, few are located among the large

rhombencephalic reticular nucleus; slh, sulcus limitans of

bers in the dorsomedial (dmc), lateral (lc), and ventral (vc)

r) of the spinal cord: cc, central canal. Scale bar represents

p e p t i d e s 2 7 ( 2 0 0 6 ) 1 0 5 4 – 1 0 7 21064

Fig. 5 – Photomicrographs showing NPFF-ir (in red) and TH-ir (in green) cells and fibers in sagittal (A, E–G) and transverse

(B–D, H) sections through the forebrain (A–G) and midbrain (H) of Petromyzon marinus. (A) General view of the lamprey

hypothalamus: AH, adenohypophysis; ch, optic chiasma; cpi, postinfundibular commissure; NH, neurohypophysis; rm,

mammillary recess. (B) NPFF-ir (in red) and TH-ir (in green) CSF-contacting cells in the bed nucleus of the tract of the

postoptic commissure (arrow) and the paracommissural preoptic nucleus (arrowhead). (C) NPFF-ir (in red) and TH-ir

p e p t i d e s 2 7 ( 2 0 0 6 ) 1 0 5 4 – 1 0 7 2 1065

labeled fibers separated by a smaller portion moderately

innervated, and a caudal part richly innervated (Fig. 4F).

Another important aspect of the NPFF-ir fibers in the lamprey

isthmus is the presence of a thin but well delineated tract of

labeled fibers that originated from the NPFF-ir cells located in

the dorsal isthmic gray. These fibers join together in the

dorsolateral aspect of the nucleus, just in front of the

emergence of the sensory and motor trigeminal roots. Then,

the fibers coursed ventromedially to reach and cross the

ventral midline through a discrete commissure located above

the heavily immunoreactive neuropil of the interpeduncular

nucleus (Fig. 1H and I). Thereafter, it was not possible to

determine the location of the target areas or nuclei of these

fibers, but it seems that they turn to constitute a diffuse

longitudinal tract together with other descending labeled

fibers from the hypothalamus.

In the rhombencephalon proper, there are several aspects

of the innervation by NPFF-ir fibers that deserve a comment.

Most of the labeled fibers were distributed throughout the

whole basal plate, whereas, labeled fibers in the alar plate

were very scarce (Fig. 1I–N). In the alar plate, a fine network of

thin immunoreactive fibers extended over all octavolateral

nuclei, with some more fibers at the border of the insertion of

the tela choroidea that covers the fourth ventricle, as well as

inside the descending trigeminal tract (Figs. 1I–N, 3D and E). In

the basal plate, the labeled fibers extended over most of the

tegmentum, but they were more abundant laterally at the

border with the descending trigeminal tract (Fig. 1I–N). Of

interest is the remarkable labeled periventricular dense fiber

plexus of the rhombencephalic branchiomotor column,

including the trigeminal, the facial, the glossopharingeal

and the vagal motor nuclei (Figs. 1I–N, 3B, C, F and 4G).

3.2.6. Spinal cordIn the spinal cord, labeled fibers were found in the lateral

columns, with the highest density at the border with the

dorsal columns, and, to a lesser extent, in the ventral and

dorsal columns (Figs. 1O, 3H and 4H). Part of them constituted

a caudal continuation of the longitudinal tracts in the

rhombencephalon, whereas, others originated from local

positive cells. In the dorsal column, labeled fibers were more

numerous ventromedially. Some thin and varicose fibers were

also seen between the motoneurons, inside the spinal motor

column (Fig. 4H).

3.3. Double NPFF and TH immunohistochemistry

The pattern observed for the distribution of NPFF immunor-

eactivity in the sea lamprey brain pointed to the possibility

of extensive codistribution with catecholaminergic cells

[62,59,60,84]. Both NPFF-ir and TH-ir cells were found in the

(in green) cells in the caudal part of the bed nucleus of the tract

(E) Part of the hypothalamus showing NPFF-ir cells (in red) in its

partial overlapping in the caudal part of the bed nucleus of the

commissure; rm, mammillary recess. (F) Detail of the square in

commissure; PVO, hypothalamic periventricular organ. (G) NPFF

neurohypophysis (NH). (H) NPFF-ir (in red) and TH-ir (in green) f

midline (arrow). Scale bar represents 500 mm (A), 100 mm (B, C,

commissure of the paraterminal preoptic nucleus (nucleus of

the anterior commissure [62]), the small cells of the magno-

cellular preoptic nucleus, the paracommissural preoptic

nucleus (a part of the nucleus of the postoptic commissure

in Pierre et al. [59,60], and Pombal and Puelles [65]), the rostral

part of the bed nucleus of the tract of the postoptic

commissure (another part of the nucleus of the postoptic

commissure in Pierre et al. [59,60], and Pombal and Puelles

[65]), the caudal hypothalamus, the rostral rhombencephalon,

the nucleus of the solitary tract, and the spinal cord. However,

in double fluorescent immunolabeled sections it became clear

that there is a high degree of codistribution of these two

substances, but colocalization in the same neuron was absent.

Both types of immunoreactive cells were clearly interspersed

in the preoptic area, including the commissure of the

paraterminal preoptic nucleus, the small cells of the magno-

cellular preoptic nucleus, and the paracommissural preoptic

nucleus (Fig. 5A and B), as well as in the rostral part of the bed

nucleus of the tract of the postoptic commissure (Fig. 5B). More

caudally in the hypothalamus, some cells of the caudal part of

the bed nucleus of the tract of the postoptic commissure

(Fig. 5C and D) and the tuberommamillary nucleus (Fig. 5E and

F) codistributed with catecholaminergic cells rostrally

migrated, whereas, in the hypothalamic periventricular organ,

and the mammillary and retromammillary regions (Fig. 5A, E

and F) only TH-ir cells were observed. Unfortunately, an

assessment of colocalization of NPFF and TH-ir cells in either

the isthmic region, the nucleus of the solitary tract, and the

spinal cord was not possible because of the absence of

immunolabeling with the anti-TH antiserum used in this

study. In addition, there was a high overlapping of labeled

NPFF and TH-ir fibers throughout the lamprey brain and spinal

cord, such as in the striatum region, the neurohypophysis

(Fig. 5A and G), the postinfundibular commissure (Fig. 5A and

E), or the optic tectum (Fig. 5H), but colocalization of both

substances was not observed in labeled fibers.

4. Discussion

The aim of this study was to provide a detailed description of

the topographical localization of NPFF-ir structures in the

central nervous system of lampreys, which actually repre-

sents the first study on the distribution pattern of this

neuropeptide in fishes. The study was performed in adult P.

marinus by using an antiserum against bovine NPFF. Our

results in the CNS of upstream-migrating adult P. marinus

show the presence of NPFF-ir cells mainly in the hypothala-

mus and the isthmic region. The comparative analysis of the

distribution of NPFF-ir structures reported herein with those

previously reported in agnathans for FMRFamide [19,54],

of the postoptic commissure. (D) Detail of the square in C.

rostral part and TH-ir cells (in green) in its caudal part, with

tract of the postoptic commissure: cpi, postinfundibular

(E). nTPOC, bed nucleus of the tract of the postoptic

-ir (in red) and TH-ir (in green) fibers in the

ibers in the optic tectum, with some crossing at the caudal

G, H), 50 mm (D), and 200 mm (E).

p e p t i d e s 2 7 ( 2 0 0 6 ) 1 0 5 4 – 1 0 7 21066

Table 1 – Comparative analysis of the cell populations immunoreactive for NPFF, FMRF, and NPY in lampreys

PTP SHL PMg AB nTPOC TM Th mt dig arn mrn ndV ola nts Spinal cord

NPFF (1) + � + + + + � + + + � + � + +

FMRF (2) � � � � + � � � + � � � � � �FMRF (3) � � � � + � � � � � � � � � �NPY (4) � + + + + � + + � + + � + � +

NPY (5) * + � + + � + * * + + * + * +

The presence (+) or absence (�) of immunoreactivity (ir) is indicated in the different populations obtained from the results in (1) Petromyzon

marinus, present study, (2) Ichthyomyzon unicuspis [19], (3) Lampetra japonica [53], (4) Lethenteron japonica [17], and (5) Lampetra fluviatilis and/or

Ichthyomyzon unicuspis [7,11,56,64,77]. The asterisk (*) indicates populations that were not studied. Abbreviations: AB, anterobasal nucleus; arn,

anterior rhombencephalic reticular nucleus; dig, dorsal isthmal gray; mrn, medial rhombencephalic reticular nucleus; mt, mesencephalic

tegmentum; ndV, nucleus of the descending trigeminal tract; nTPOC, bed nucleus of the tract of the postoptic commissure; nts, nucleus of the

solitary tract; ola, octavolateral area; PMg, magnocellular preoptic nucleus; PTP, paraterminal preoptic nucleus; SHL, subhippocampal lobe; Th,

thalamus; TM, tuberomammillary nucleus.

and/or neuropeptide Y (NPY) [11,17,65,79,88] showed marked

differences (see Table 1 and Section 4.2), suggesting that a

NPFF-like peptide exists in lampreys. Finally, the relationship

of the distribution of NPFF-ir to catecholaminergic (TH-ir)

cells was analyzed by using a double immunohistochemistry

technique.

4.1. Comparison of NPFF-ir structures in the lampreybrain with previous results in amphibians and mammals

In the following paragraphs, we compare our findings on the

distribution of NPFF-immunoreactivity in the lamprey CNS

with those previously reported on several mammalian species

[33,34,42,45,46,55,56], as well as in some amphibians (Rana

perezi, Xenopus laevis, and Pleurodeles waltl; [18].

4.1.1. TelencephalonIn this research, no NPFF-ir cells were found in the olfactory

bulbs or the telencephalon of the adult sea lamprey. Labeled

cells in these two brain subdivisions have only been detected

in amphibians [18]. Concerning the NPFF-ir fibers, the

distribution pattern in lampreys with scattered fibers in the

olfactory bulbs and pallial subdivisions, and numerous fibers

in the subpallium, including the septum and the striatum, is

more similar to that reported in amphibians [18] than that

found in mammals, where the cortical regions and the basal

ganglia are almost free of labeling or poorly innervated

[33,46,55]. More studies are needed covering a more diverse

range of vertebrates before determining whether these

differences represent an evolutionary change between ana-

mniote and amniote vertebrates.

Finally, very few NPFF-ir fibers were present in the optic

nerve. In this nerve, occasional fibers were also immunoreac-

tive for either GnRH or FMRFamide [19]. A possibility raised by

these authors, is that these fibers could be homologous with

the retinopetal component of the terminal nerve system that

has been characterized in many other vertebrates (e.g., [51,86]).

Curiously enough, there is the presence of NPFF-ir cells in the

ganglion of the terminal nerve in both Carassius auratus [40],

and Pleurodeles waltl [18] with the same antibody used in this

study. In addition, a NPFF-related gene recently cloned in

zebrafish (Danio rerio) is selectively expressed only in neurons of

the terminal nerve [53]. However, based on experimental

material regarding the organization of the retinal projections

and immunohistochemistry for those substances reported in

other species, the existence of a terminal nerve in lampreys is

still very controversial (see [19,82]).

4.1.2. HypothalamusAs in mammals and amphibians, this study reveals that most

of the NPFF-ir cells in the lamprey brain are located in the

hypothalamus, including distinct subdivisions of the preoptic,

tuberal, and mammilar regions. In amphibians there are NPFF-

ir cells in the suprachiasmatic nucleus, the retrochiasmatic

nucleus, and the anterodorsal portion of the infundibulum of

anurans, whereas, in urodeles they are located in the anterior

preoptic area, the suprachiasmatic nucleus, and the dorsal

aspect of infundibular hypothalamus [18]. In mammals most

NPFF-ir cells were initially located between dorsomedial and

ventromedial hypothalamic nucleus (i.e., in the intermedio-

medial nucleus; see [55]); but some were also reported in the

supraoptic and paraventricular nuclei [6,31]. However, it is

now known that the NPFF immunoreactivity of the inter-

mediomedial nucleus in mammals is due to other peptide

belonging to the NPVF family of peptides [43,76]. Although no

definitive comparisons between species concerning labeled

nuclei can be made, some similarities as well as differences

appear to exist. As an example, the neurosecretory neurons of

the paraventricular nucleus of rats have been shown to

contain both NPFF and vassopressin and to project to the

neurohypophysis [49], where both substances colocalize in

nerve terminals as visualized by post-embedding immunoe-

lectron microscopy [41]. However, the paraventricular nucleus

of lampreys that contains vasotocin (the lamprey counterpart

of vassopressin; [65]) and projects to the neurohypophysis (see

[63,64]) was not immunoreactive for NPFF. There are numer-

ous vasotocin immunolabeled cells projecting to the neuro-

hypophysis in the magnocellular preoptic nucleus [22,65], and

only the small NPFF-ir cells of this nucleus could contain

vasotocin, which seems highly unlikely. However, the NPFF-ir

cells in the dorsal infundibular portion of the amphibian

hypothalamus [18], and the intermediomedial nucleus of

mammals [33,34,35,37,42,56], could be considered equivalent,

at least in part, to the bed nucleus of the tract of the postoptic

commissure and/or the tuberomammillary nucleus of lam-

preys. Some correspondence could be established between the

labeled cells located in the preoptic and supraoptic areas of

lampreys, amphibians, and mammals.

p e p t i d e s 2 7 ( 2 0 0 6 ) 1 0 5 4 – 1 0 7 2 1067

Concerning the NPFF innervation, the hypothalamus of

amphibians and mammals are richly innervated by labeled

fibers [18,41,49]. The lateral walls of the lamprey hypothalamus

are also innervated by a conspicuous fiber plexus, which

partially enters the neurohypophysis. In the hypophysis of

amphibians, the intermediate lobe is primarily innervated by

NPFF-ir fibers [18], whereas in rat labeled fibers enter the neural

lobe (median eminence [6,31,41,49]. In lampreys, the anterior

neurohypophysis has been compared to the mammalian

median eminence, whereas, the posterior neurohypophysis

would correspond to the neural lobe (see [74]. Although not

evenly distributed, our results demonstrated that both

parts are highly innervated by varicose fibers; therefore,

this peptide may be involved in hypothalamic regulation

of pituitary function in lampreys as previously shown in

mammals [47,49].

4.1.3. DiencephalonIn the sea lamprey diencephalon, NPFF-ir cells were only

present in the parapineal vesicle with a rich fiber innervation

in its ganglion. This is the first time that some NPFF

immunoreactivity is reported in the epiphysis of vertebrates.

The bipolar shape of the labeled cells in the ventral wall of the

parapineal vesicle suggests that the cells correspond to

photoreceptors. These cells give rise to nerve terminals in

the parapineal ganglion, as well as to some fibers running in

the parapineal tract, and thus appear to form part of both

local and external circuits. In a previous study cells immu-

noreactive for choline acetyltransferase (ChAT-ir) were

reported in the ventrocaudal region of the parapineal vesicle

[90]. Although the distribution of both NPFF-ir and the

ventrocaudal ChAT-ir cell group is similar, they probably

represent separated populations on the basis of the different

thickness and shape of cells; however, this should be

confirmed by using double labeling. Apart from the labeled

cells of the lamprey parapineal, NPFF-ir cells have been also

found in the central nucleus of the dorsal thalamus of

Xenopus [18].

4.1.4. MesencephalonIn this study of the lamprey, we found a single cell population

of NPFF-ir neurons in the ventromedial mesencephalic

tegmentum. It is not clear as to whether or not the labeled

cells belong to the M5 nucleus of Schober, where some of the

retinopetal cells are located in these animals (see [80] for a

review). If it is assumed that the NPFF-ir cells correspond with

the retinopetal cells, then the presence of some labeled fibers

in the optic nerves could represent the axons of these cells on

the way to the retina. A similar population was previously

reported in the mesencephalon of Xenopus, but located

laterally [18]; however, in amphibians there are no retinopetal

cells located in the mesencephalon (see [75] for a review).

Therefore, the labeled cells in the lamprey could correspond to

or be homologous of those reported in Xenopus. In lamprey,

tachykinin-ir cells [4] were also reported in a similar location.

The presence of numerous NPFF-ir fibers in either the

mesencephalic tegmentun and optic tectum of the lamprey

brain is more similar to what was reported in amphibians [18]

than in mammals, where the NPFF innervation was scarce or

absent [33,55].

4.1.5. RhombencephalonThe isthmus of lampreys possesses numerous NPFF-ir cells

located in two populations, one located in the dorsal isthmal

gray and other belonging to the reticular formation. However,

no labeled cells were previously found in the isthmus of the

other species of vertebrates studied. Of interest is the

existence of abundant labeled fibers originated from the

positive isthmic cells of lampreys that appears to innervate

the ventromedial interpeduncular neuropil, as well as to joint

the rostrocaudal fiber tracts that run up to the spinal cord. An

extremely intense longitudinal fiber tract from the level of the

isthmus to the spinal cord was shown in urodele amphibians

[18]. The origin of this tract is unclear. One possibility is that

the cells are located in the dorsal portion of the isthmus, but

cannot be distinguished because of the high concentration of

labeled fibers in such region.

Many NPFF-ir cells were present in the rhombencephalic

reticular formation of the lamprey, but restricted to the

anterior rhombencephalic reticular nucleus. Immunolabeled

reticular cells have been shown also in the median and inferior

reticular formation of anurans, but not in urodele amphibians

[18]. Conversely, in the reticular formation of the mammals

studied so far, no labeled NPFF-ir cells were found.

The immunopositive neurons located in the trigeminal

sensory nucleus of lampreys are not evenly distributed

throughout the nucleus but occupy the rostral one third,

where sensory information carried by fibers of the five

branches of the trigeminal nerve (i.e., the ophthalmic, apical,

basilar, suborbital, and mandibular) appears to be processed

[39]. However, the location of the NPFF labeled cells, i.e., the

ventral part of the descending trigeminal tract, better

corresponds with the distribution area of fibers from the

ophthalmic branch [39]. In rats, a strong signal for NPFF mRNA

was found in the spinal nucleus of the trigeminal nerve by in

situ hybridization [81].

The motor neurons of the branchial nerves themselves also

appear to receive an NPFF innervation. The branchiomotor

column of lampreys (including the trigeminal, the facial, the

glossopharingeal and the vagal motor nuclei) consists of a

more or less continuous band of neurons along the rhom-

bencephalon (see [64]). The density of NPFF positive fibers is

relatively low around the neuronal somata, but moderate on

the ventrolateral aspect of the cell column and high on its

periventricular margin, where the main dendrites of the

motoneurons extend. In mammals, NPFF has been implicated

in sensory systems, most notably pain and morphine

analgesia [88]. The location of the immunopositive cell bodies

in the trigeminal sensory nucleus and the presence of a

terminal arborization around the branchiomotor column

suggest the NPFF plays a role in the sensory-motor control

of the branchial apparatus, even though many details of this

interaction remain obscure.

The presence of NPFF-ir cells in the nucleus of the solitary

tract is a common feature of all vertebrates studied so far,

including lampreys [18,33,56]. In addition, in rats some of the

neurons, which are positive for NPFF are also positive for

tyrosine hydroxylase [32,42]; see also [55]. Although some

neurons have been shown to be immunoreactive for dopa-

mine in a location considered in lampreys to be homologous to

the nucleus of the solitary tract [62], the anti-TH monoclonal

p e p t i d e s 2 7 ( 2 0 0 6 ) 1 0 5 4 – 1 0 7 21068

antibody used in this study did not label any cells in that

position (see Section 4.3). Therefore, it cannot be confirmed

whether or not both substances coexist in the same cells of

this population.

4.1.6. Spinal cordSome NPFF-ir cells have been detected in the central gray

matter of the lamprey spinal cord. Weakly labeled cells in a

similar location were found in amphibians [18], which have

been suggested to be comparable to those identified in the

spinal cord of the rats [36]. The innervation of the spinal cord

by NPFF-ir fibers is more conspicuous in the dorsal part,

particularly in the dorsal horn, in all species studied so far

[18,21,36,45]. Although experiments with tracing techniques

suggest that NPFF immunoreactivity in the spinal cord of rats

is of intrinsic origin [36], some contribution from a descending

tract has been suggested [21,45]. Although there are some

labeled cells in the spinal cord of lampreys with their

corresponding processes, most of the fibers innervating the

spinal cord constituted a caudal continuation of the long-

itudinal tracts found in the rhombencephalon. This is

consistent with results obtained in amphibians [18], where

the innervation of the spinal cord by descending fibers forming

a strong immunoreactive tract is unequivocally distinguished

in urodeles.

4.2. Comparison with other RFamide and RYamide-relatedpeptides reported in the lamprey brain

The distribution pattern of NPFF-ir cell bodies and fibers in the

lamprey brain shows many similarities to those of other

neuropeptides (for review see [52]). Furthermore, some

polyclonal antisera to FMRF-amide (a member of the RFa-

mide-related peptides: FaRPs) or NPFF have been found to

weakly cross-react with neuropeptide Y (NPY, a member of the

RYamide-related peptides) [46,67]. However, a comparative

analysis of the distribution of these neuropeptides to that of

NPFF in lampreys showed marked differences (see Table 1)

pointing to a lack of cross-reactivity of the antiserum used in

this study with those neuropeptides.

In this section, we discuss our results concerning the

labeling found in agnathans with antisera against FMRFamide

and NPY noting that cross-reactivity of our polyclonal anti-

body for NPFF with these peptides could also be expected.

Previous studies have reported the distribution of FMRFamide-

like immunoreactivity (FMRF-ir) using different antisera in the

agnathan brain, including three hagfish species: Eptatretus

burgeri [28], Eptatretus stouti [85], and Paramyxine atami [15], and

two species of lamprey: Lampetra japonica [54] and Ichthyomuzon

unicuspis [19]. In addition, some FMRFamide-ir fibers were also

reported in the spinal cord of Lampetra fluviatilis [12]. Some

immunohistochemical studies have provided basic data on the

organization of NPY-like-immunoreactive (NPY-ir) structures

in the brain of cyclostomes, including a hagfish (Paramixine

atami: [14]), and several species of lampreys (Lampetra fluviatilis

and/or Ichthyomyzon unicuspis: [7,11,57,65,79], and Lethenteron

japonica: [17]). In addition, the identity of the peptide in lamprey

has been verified with in situ hybridization [69].

Most immunoreactive cells for these two substances in the

lamprey brain were found in the preoptic and infundibular

hypothalamus. Some small NPY-ir cells were observed in the

subependymal layer of the preoptic area [17]. These cells could

correspond to those NPFF-ir cells found in this research to be

located in the same position. A conspicuous population of

NPY-ir cells was found in the dorsal hypothalamus [17,65].

Most of the FMRF-ir cells were also found in the dorsal

hypothalamus [19,54]. These hypothalamic cells, that were

initially subdivided in three groups (‘‘rostral’’, ‘‘dorsal’’ and

‘‘caudal’’) by Ohtomi et al. [54], and later confirmed by Eisthen

and Northcutt [19], roughly correspond to our anterobasal

nucleus plus bed nucleus of the postoptic commissure. While

this is the only location in the lamprey brain where positive

cells for the three peptides were found; the putative coex-

istence in the same cell should be further explored with double

labeling experiments. As has been shown previously, the cells

initially reported as NPFF-ir in the hypothalamic intermedio-

medial nucleus of rats do not store NPY [35].

In hagfish FMRF-ir cells were almost restricted to hypotha-

lamic structures, including the infundibular hypothalamic

nucleus [15,28,85], and, occasionally, in the surrounding areas.

In addition, NPY-ir cells were also found in the same region in

these animals, and the examination of adjacent sections

immunostained alternatively for NPY and FMRFamide showed

overlapping of the immunoreactive structures, with the same

cells immunostained with both antisera. However, it should be

noted that cross-blocking experiments showed that FMRF-ir

structures could be recognized by the anti-NPY antiserum they

used. In lamprey, there are some other cell populations

immunoreactive only for NPY, such as the dorsal pallium (our

subhippocampal lobe), the preoptic area, the ventral part of

the dorsal thalamus, and the juxtacommissural pretectum

[17,65,90]. Furthermore, in the medial portions of the

mesencephalic tegmentum there are some NPY-ir cells

[11,17] in the same location where NPFF-ir cells were detected.

In the lamprey rhombencephalon, some weakly and

scattered FMRF labeled cells were also seen, in two out of

five animals, in the dorsal isthmal gray [19], which clearly

corresponds to the NPFF-ir dorsal isthmic group we found.

In this study, NPFF-ir cells were also found in the trigeminal

sensory nucleus. FMRF-ir cells were reported in the trigeminal

sensory nucleus of hagfish [85]; however, none were present in

lampreys [19,54]. Concerning the reticular formation, NPY-ir

cells were described in the middle rhombencephalic reticular

nucleus [17]. However, we consider that the NPY-ir cells found

by Chiba [17] in caudal mesencephalic tegmentum (see his

Fig. 2G) to correspond to our anterior rhombencephalic

reticular nucleus (see also [11]). Therefore, NPY-ir cells are

present in both the anterior and the middle rhombencephalic

reticular nuclei, whereas, NPFF-ir cells were only observed in

the caudal part of the anterior rhombencephalic reticular

nucleus. Additional NPY-ir cells were reported in the medial

portions of the rhombencephalic alar plate from the isthmic

region up to the level of the vagal motor nucleus [11,17]. Part of

these cells in the caudal rhombencephalon could correspond

to the nucleus of the solitary tract in the lamprey. Experiments

in the same nucleus of rats demonstrated that neurons

immunoreactive for NPFF and NPY comprise distinct popula-

tions of cells [35].

In the spinal cord of lampreys, FMRFamide-ir cells were not

reported [12,19,54]. However, some NPY-ir cells were observed

p e p t i d e s 2 7 ( 2 0 0 6 ) 1 0 5 4 – 1 0 7 2 1069

in the dorsal part of the cell column [7,11,17,57,79]. In addition,

the NPY-ir fibers appeared to originate from these cell bodies

and were restricted to the dorsal half of the spinal cord [11,79].

Because of NPFF-ir was occasionally seen in the same fibers

with NPY in the porcine spinal cord [83], these substances

could also colocalize to the same neurons. Double labeling

techniques are needed to test this hypothesis.

Fibers immunoreactive for FMRFamide did not form

discrete bundles, but were scattred diffusely throughout

diverse regions of the brain and rostral spinal cord

[12,19,54]. Concerning the neurohypophysis, labeled fibers

were observed in Ichthyomyzon unicuspis [19], but only present

to a limited extend in its anterior part (median eminence) in

Lampetra japonica [54]. In addition, no FMRFamide immunor-

eactivity was found in the neurohypohysis of the three species

of hagfish studied so far [15,28,85]; however, there is a rich

innervation by NPFF-ir fibers. Thus, whereas, the hypotha-

lamic immunoreactive cells for FMRF do not participate in

neuroendocrine mechanisms in hagfishes and their role in

lampreys appears to be very limited, those labeled for NPFF

appear to be highly involved in the regulation of the

neurohypophysis by the hypothalamo-hypophyseal system

(see Section 4.1.2).

4.3. Codistribution of NPFF and catecholamines

In the present study, the presence of NPFF in the catechola-

minergic cells has been investigated, since these substances

are present in neurons that show similar distribution patterns

[59,60,62,84]. If NPFF colocalizes with the catecholamine-

synthesizing enzyme TH, it could act as a cotransmitter in

catecholaminergic neurons and thus be involved in various

somatosensory and visceral functions. Although the NPFF-ir

cells were found in several places where interaction with

catecholaminergic cells could be expected, no colocalization

was found throughout the sea lamprey brain and spinal cord.

However, our findings are of significance in that they add

confirmation to the previously reported subdivisions of the

lamprey preoptic area and hypothalamus [65]. In addition, our

study showed an extensive codistribution of NPFF-ir and TH-ir

fibers in several regions or nuclei in the lamprey CNS. With

this technique, the TH-ir structures (green) and the NPFF-ir

structures (red) are well distinguished in separate neurons.

However, sometimes they appeared somehow masked (show-

ing a yellow-like color), particularly in those areas of high

concentration of labeled fibers such as the neurohypophysis or

the postinfundibular commissure (see Fig. 5A). This is

certainly due to overlapping of fibers with different immunor-

eactivity (TH-ir or NPFF-ir) because of the thickness of the

sections (40 mm), but not because of colocalization of both

substances (TH and NPFF) in the same fiber.

Unfortunately, the presence of double labeled cells for NPFF

and TH in either the isthmic region, the nucleus of the solitary

tract, and the lateral spinal cord could not be studied because

of the absence of immunolabeling with the monoclonal anti-

TH antiserum used in our study. This was somehow expected

because the presence of catecholaminergic cell bodies in these

populations as revealed by using antisera against TH and/or

dopamine has been difficult to demonstrate in lampreys

[59,60,62]. As an example, all the anti-TH antibodies used

previously failed to detect any labeling in the nucleus of the

solitary tract and the lateral spinal cord [59,60,62], whereas,

some labeling was reported in both by using a non-

commercial anti-dopamine antibody [62,68].

5. Conclusions

The comparative analysis of the distribution of FMRF-ir and/

or NPY-ir with that of NPFF-ir structures in the lamprey CNS

showed a partial overlapping which suggests that a NPFF-

like peptide also exists in this animal. Furthermore, the NPFF

immunoreactive network of fibers appears to be well

developed in the brain of the lamprey, a living member of

the most primitive vertebrates. This study reveals that, in

general, the lamprey pattern of NPFF-ir elements is similar to

that previously reported for other vertebrates. In addition, the

organization of some of the NPFF-ir systems in the lamprey

CNS as, for example, the innervation of the neurohypophysis or

the spinal cord, comprises features of the anamniote (amphi-

bian) brain that are also common to all amniotes (mammals)

studied so far, thus being conservative to a surprisingly high

degree. Although more studies are needed, this also suggests

that the function of this neuropeptide could be conserved

through vertebrate evolution. Finally, the double immuno-

fluorescent experiments have shown that, in spite of the high

degree of codistribution found, colocalization of NPFF and TH

was absent in all labeled cell populations.

Acknowledgements

We want to express our gratitude to Dr. H.-Y.T. Yang for his

generous gift of the antiserum used in this study, and Dr. S.

Nagi for copy-editing suggestions. We also thank the Con-

sellerıa de Medio Ambiente of the Xunta de Galicia for their

kind permission to capture of the lampreys used in this study,

as well as M. Garcıa, J. Garcıa and J.A. Garea at the Center for

Control of Fishes of Ximonde (A Coruna). This study was

supported by grants from Ministerio de Ciencia y Technologıa-

FEDER (BFI2003-04479), Xunta de Galicia (PGIDT04P-

XIC31004PN), and University of Vigo (Z441-64102) to MAP,

and Ministerio de Ciencia y Tecnologıa (BFI2003-0559) to AG.

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