Comparative distribution of urocortin- and CRF-like immunoreactivities in the nervous system of the earthworm Lumbricus terrestris

Peptides 24 (2003) 205–213

Comparative distribution of urocortin- and CRF-like immunoreactivities in the nervous system of the earthworm Lumbricus terrestris Andrea Lubics a,∗ , Dóra Regl"odi a,b , Márta Szelier a , István Lengvári a , Tamás Kozicz a a

Department of Human Anatomy, Medical Faculty, University of Pécs, Szigeti ut 12, Pécs 7624, Hungary b Neurohumoral Regulation Research Group of the Hungarian Academy of Sciences, Medical Faculty, University of Pécs, Szigeti ut 12, Pécs 7624, Hungary Received 12 September 2002; accepted 6 November 2002

Abstract Corticotropin-releasing factor (CRF) and urocortin (Ucn) are both members of the CRF neuropeptide family. The distribution of Ucnand CRF-like immunoreactive (ir) structures in the central nervous system of several vertebrate species has been studied, but little is known about that in non-vertebrates. We used a highly specific polyclonal antibody against rat Ucn and CRF to determine and compare the distribution of Ucn- and CRF-like immunoreactivity in the earthworm nervous system. Several Ucn- and CRF-like ir perikarya were described in the cerebral ganglion, subesophageal and ventral cord ganglia. The majority of Ucn-like ir cells were found in the ventral ganglia, whereas CRF-like ir cells were most abundant in the cerebral ganglion. Scattered Ucn- and CRF-like ir varicose fiber terminals were seen in all areas of the earthworm central nervous system. Ucn-like ir cell bodies and fiber terminals were also demonstrated in the pharyngeal wall. No co-localization of Ucn- and CRF-like ir nervous structures were observed. This study provided morphological evidence that Ucn- and CRF-like neurosecretory products exist in the earthworm central nervous system. Furthermore, both the distribution and morphology of Ucn- and CRF-like ir structures were distinct, therefore, it can be hypothesized that these neuropeptides exert different neurendocrine functions in the earthworm nervous system. © 2003 Elsevier Science Inc. All rights reserved. Keywords: Urocortin; CRF; Annelid; Earthworm; Nervous system; Immunohistochemistry

1. Introduction Corticotropin-releasing factor (CRF), a 41 amino acid containing neuropeptide was first isolated from the mammalian brain, and it is the major coordinator of endocrine responses to stress mainly through a neurohormonal action on the hypothalamo–pituitary–adrenal axis [11,35]. It also serves as a neurotransmitter, thereby integrating autonomic and immunological responses to stress [35]. Furthermore, CRF has also been found to be involved in regulation of food intake [3]. Another CRF-related neuropeptide, urocortin (Ucn), that is structurally and pharmacologically similar to CRF, was discovered by Vaughan et al. in 1995 [36]. Substantial neurobiological evidence points to an important physiological and endocrine role for Ucn in the central nervous system [11,19,34]. The distribution of CRF in both vertebrate and nonvertebrate species has been thoroughly studied [26,32,38]. ∗ Corresponding author. Tel.: +36-72-526-000x1828; fax: +36-72-536-393. E-mail address: [email protected] (A. Lubics).

Similarly to CRF, Ucn is widely distributed in the mammalian central nervous system and in various peripheral organs [21,22,27]. In general, the distribution of Ucn and CRF in different vertebrates shows very little neuroanatomical overlap, suggesting different physiological significance for these neuropeptides [27]. A number of vertebrate neuropeptides have been described in the invertebrate Annelida [1,10,18,31]. Thus, the distribution of CRF-like immunoreactivity has also been demonstrated in a few invertebrate species, such as in insects [33,37,38] and in one annelid species [32]. However, the distribution of Ucn-like immunoreactivity has not been studied in invertebrates yet. One of the most widely used invertebrate models is the phylum of Annelida. The annelid central nervous system contains a relatively small number of neurons, and besides having a segmentally arranged ventral cord, centralization first occurs in this phylum during phylogeny in form of the appearance of a cerebral ganglion [6]. Investigation of the invertebrate nervous system is important for elucidating evolutionary questions, and it may also provide a better understanding of basic neuronal mechanisms. Therefore, the

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aim of this study, using highly specific polyclonal antibodies against CRF and Ucn, were to examine and compare the distribution of Ucn and CRF in the earthworm (Lumbricus terrestris) nervous system.

2. Materials and methods 2.1. Animals Adult specimens of the earthworm L. terrestris were purchased from a local supplier. Ten earthworms were used in our experiments. All procedures were conducted according to the Law of 1998. XXVIII for Animal Care and Use in Hungary and in accordance with the animal use guidelines approved by the Medical Faculty Advisory Committee for Animal Resources, Pécs University. 2.2. Colchicine treatment Four worms received colchicine treatment in order to increase the amount of immunoreactivity in neuronal cell bodies, whereas six were not treated. The crystalline colchicine (Sigma Chemical Co., St. Louis, MO) was dissolved in 0.9% saline at a final concentration of 0.2 ␮g/␮l. Each animal received 1 ␮g of colchicine. The survival time was 24–36 h. 2.3. Fixation The segments anterior to the clitellum were removed and fixed in picric acid (1.5%)–formaldehyde (1%) fixative. Penetration of the fixative was promoted by treating the specimens with microwave irradiation (750 W, 10 s, 10 ml ice-cold fixative). Excess amount of fixative was removed by washing the samples in phosphate-buffered saline solution (PBS). 2.4. Antisera used in this study The rat Ucn antiserum was a gift from Dr. Wylie Vale (The Salk Institute for Biological Studies, La Jolla, CA, USA). This antibody (PBL #5779) was generated in rabbit against rat Ucn coupled to human ␣-globulins via glutaraldehyde as immunogen. The antiserum was previously used and characterized in several studies [5,23]. The CRF antiserum was gift of Dr. Akira Arimura (Tulane University, US–Japan Biomedical Research Laboratories, New Orleans, LA, USA), and was previously characterized in his laboratory [20]. 2.5. Controls 2.5.1. Anti-Ucn antiserum (#5779) In the control experiments, a 1:300,000 dilution of the primary antiserum was optimal for immunostaining. Increasing the dilution of the primary antiserum decreased

the immunoreaction until a 1:1,000,000 dilution, at which point no immunostaining was visible. The specificity of the primary antiserum was further tested on earthworm brain sections by adsorption of the primary antiserum with the synthetic peptide (Ucn) used for immunization or other structurally-related peptides (CRF, urotensin I and sauvagine). Competition studies showed that the synthetic immunogen Ucn at a concentration as low as 1 ␮g/ml dramatically attenuated staining in sections from the earthworm brain, whereas 10 ␮g/ml of the synthetic immunogen completely abolished the immunostaining. The two closely-related family members human/rat CRF and sauvagine did not affect the immunostaining in earthworm brain sections at a concentration as high as 100 ␮g/ml. The closest known family member urotensin I did not noticeably affect staining at 10 ␮g/ml and attenuated the immunostaining only slightly at a concentration as high as 100 ␮g/ml. Qualitatively similar adsorption results have been reported with the same antiserum and sections from the rat [5] and the frog brain [23]. 2.5.2. Anti-CRF antiserum (#86/294) In immunohistochemical tests, the immunoreaction was completely blocked after the antiserum was preincubated with 5, 25 and 50 ␮g/ml of synthetic CRF. Preincubation with 5, 25 and 50 ␮g/ml of synthetic Ucn did not alter immunostaining. When the primary antibodies were omitted or replaced by non-immune rabbit sera at a dilution of 1:30,000 for Ucn or 1:5000 for CRF, no immunoreaction was observed. 2.6. Immunohistochemical procedure Immunohistochemistry was performed on serial frontal cryostat (Leica, Nussloch, Germany) sections (10 ␮m) mounted on slides coated with gelatine for better adhesion. The sections were treated with trypsin solution (0.12%, Reanal, Budapest). Unspecific background staining was inhibited by incubation in 1% normal sheep serum (NSS) (20 min). The sections were stained according to the unlabelled antibody enzyme peroxidase-antiperoxidase (PAP) method, similarly to our previous studies [31]. The polyclonal anti-Ucn (diluted 1:300,000 in PBS) or the polyclonal anti-CRF (diluted 1:5000) was applied for 48 h at 4 ◦ C. The rest of the staining procedure was carried out at room temperature. The sections were then incubated with anti-rabbit ␥-globulin (ARGG, sheep, 1:200) for 10 min, and PAP (1:500) for 10 min. ARGG was raised in our laboratory and PAP was obtained from Arnel (New York, NY, USA). The entire procedure starting from the treatment with ARGG was repeated (double PAP method). Thorough washing in PBS was performed after each step of staining procedure. Bound PAP was visualized by 0.25% 3,3-diaminobenzidine in 0.05 M Tris buffer at 7.5 pH containing 0.01% H2 O2 (20 min). The diaminobenzidine end product was intensified in osmium tetroxide vapor. For the co-localization studies,

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serial frontal sections (10 ␮m) from three animals were alternately mounted on separate slides. 2.7. Immunofluorescence procedure The immunofluorescence studies were also performed on serial frontal cryostat sections treated with trypsin solution. Unspecific background staining was inhibited by incubation in 5% normal donkey serum (Jackson Immonoresearch Labs, West Groove, PA, USA) in PBS for 30 min to block non-specific binding sites. The sections were incubated overnight at room temperature in either a rabbit polyclonal anti-Ucn antibody diluted at 1:30,000 or polyclonal anti-CRF antibody diluted at 1:1000. After incubation in the primary antibodies, the sections were washed for 10 min in normal donkey serum followed by 3 × 10 min washes in PBS. The secondary antiserum, the rhodamine RedTM -X-conjugated anti-rabbit IgG was used at 1:80 dilution (Jackson Immunoresearch Labs, West Groove, PA, USA) dissolved in 2% normal donkey serum, and applied for 4 h at room temperature in a humid chamber. Following several washes in PBS, the slides were coverslipped with an antifade mounting medium (Vectashield, Vector Labs, Burlingame, CA, USA). 2.8. Digital imaging All photomicrographs were taken with a Nikon Microphot FXA microscope equipped with a Spot RT color digital camera. Digital images of immunolabeled sections, using software supplied with the Spot RT color camera were taken at a resolution of 1200 × 1600 pixels. The images were transferred to an IBM compatible PC and digitally processed (size and histogram levels were adjusted; images were combined into plates and figures) using Adobe Photoshop 5.5. Comparison of consecutive sections was performed on digitized images using the NIH Image 1.55 program for Macintosh. Parallel projection of adjacent sections of ganglia enabled the localization of double stained cells, as described earlier [31]. Using the “AND” image processing operation on two consecutive sections, only those immunostained cells that had the same position in both sections were seen.

3. Results In Annelida, the central nervous system consists of the cerebral ganglion, and a segmentally arranged ganglionic chain called ventral cord ganglia. The three cranial ganglia of the ventral cord are fused to form the subesophageal ganglion, which is connected to the cerebral ganglion by the circumpharyngeal connectives (Fig. 1). Immunoreactivity to Ucn and CRF antisera was found in each part of the earthworm central nervous system (Figs. 1–4). Ucn-like immunoreactivity was most intensive in the ventral ganglionic chain (Figs. 1, 2F and G), whereas

Fig. 1. Distribution pattern of CRF (left side) and urocortin (right side) immunopositive elements in the cerebral (a), subesophageal (b) and ventral cord (c and d) ganglia of Lumbricus terrestris. Co: connectivum; DL: dorsolateral; DM: dorsomedial; L: lateral; M: medial; VL: ventrolateral; VM: ventromedial cell groups; NI and NII: segmental nerves. Only the location of cells and fibers and not their exact numbers or density are given.

CRF-like immunoreactivity was most abundant in the cerebral ganglion (Figs. 1 and 3A). There was no difference in the intensity of staining between colchicine-treated and -untreated animals. 3.1. Cerebral ganglion Most nerve cells are located in the dorsal cell mantle of the cerebral ganglion, divided into medial, dorsomedial, dorsolateral and lateral cell groups. A few cells are found in the ventral and central parts of the ganglion, but these cells did not form specific cell groups [6]. Only few Ucn-like immunoreactive (ir) neurons were found in the cerebral ganglion, most of them were situated in the central part of the ganglion (Fig. 2A). In contrast,

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Fig. 2. (A) Ucn-like immunopositive elements in the central nervous system of the L. terrestris (immunofluorescens staining). Dorsomedial (arrow) and dorsolateral (arrowheads) cells showing Ucn-like immunoreactivity in the cerebral ganglion. Scale bar: 30 ␮m. (B) Ucn-like immunopositive fibers crossing the midline in the cerebral ganglion (arrowheads). Scale bar: 30 ␮m. (C) Ucn-like ir cells are located in all cell groups of the subesophageal ganglion: the arrow points to a ventromedial group of cells. Ventrolateral Ucn-like ir cells are shown by arrowhead, whereas a lateral group of somata is pointed by a double arrow. Scale bar: 50 ␮m. (D) Ucn-like immunopositive cells in the subesophageal ganglion (arrows). Small arrowheads point to Ucn-like ir fiber terminals in the central neuropil. Scale bar: 35 ␮m. (E) Ucn-like ir fibers in the circumpharyngeal connectives (arrowheads). Scale bar: 25 ␮m. (F) Ventromedial (arrow) and ventrolateral (arrowhead) cells in the ventral ganglion (these cells are shown enlarged in insert). The double arrow points to cells in the lateral part of the ganglion. Scale bar: 60 ␮m, in insert: 20 ␮m. (G) The Ucn-like immunopositive cells show an arch-like distribution in the ventral part of the ventral ganglion (arrowheads). Ucn-like ir fibers are shown by small arrowheads. Scale bar: 25 ␮m.

CRF-like ir cells were mainly observed in the dorsolateral and lateral cell groups (Fig. 3A). A few immunopositive cells were seen in medial and dorsomedial positions. At the origin of the circumpharyngeal connectives, immunoreactive cells to both antisera were demonstrated (Figs. 1 and 3A). The cerebral ganglion contains approximately 2000 neurons [2]. The CRF- and Ucn-like ir cells represented 1.5–2.5 and 0.2–0.6% of the total cell number, respectively (Table 1). There was a difference also in the morphology between the CRF- and Ucn-like ir cells. The Ucn-like ir neurons were large, measuring 15–20 ␮m, and were pyriform in shape (Fig. 2A). The CRF-like ir neurons were smaller, round or oval in shape, measuring 10–12 ␮m (Fig. 3A). In some

cases, the originating part of the axon was stained, and could be traced to the central neuropil of the cerebral ganglion. The central neuropil displayed strong immunoreactivity with both antisera, with occasional varicosities (Figs. 2B and 3F). CRF- and Ucn-like ir varicose axons were observed to cross-midline in the cerebral ganglion of the earthworm (Figs. 2B and 3F). No co-localization of Ucn- and CRF-like immunoreactivities in neuronal structures was observed in the cerebral ganglion. The circumpharyngeal connectives take their origin at the ventrolateral part of the cerebral ganglion. Most of the fibers connect the cerebral ganglion to the ventral chain, while a smaller portion runs to the pharyngeal wall through the stom-

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Fig. 3. CRF immunopositive nerve cells and fibers in the central nervous system of the L. terrestris. (A–E: DAB staining, F–H: immunfluorescens staining). (A) CRF-ir cells in the lateral part of the cerebral ganglion (arrowheads) at the beginning of the connectives (Co). Scale bar: 50 ␮m. (B and C) CRF immunoreactive cells in the lateral part of the subesophageal ganglion (arrow). The lateral cells are enlarged in insert C. Scale bar in picture B: 100 ␮m, in picture C: 20 ␮m. (D and E) Ventromedially located CRF immunopositive cells in a ventral ganglion. The arrow points to CRF-ir cells that are enlarged in insert E. Small arrowheads show immunopositive nerve fibers in the central neuropil. Scale bar in picture D: 100 ␮m, in picture E: 25 ␮m. (F) CRF immunopositive fibers crossing the midline in the central part of the brain (arrowheads). Scale bar: 50 ␮m. (G and H) Ventromedial (arrow), ventrolateral (arrowhead) and lateral (double arrow) CRF-ir somata in the ventral ganglion. Scale bar in picture G: 20 ␮m, in picture H: 30 ␮m.

Fig. 4. Ucn-like immunopositive elements in the pharyngeal wall of the L. terrestris. (A and B) Ucn-like ir fibers in the in subepithelial and submucosal (arrowheads) locations in the pharyngeal wall. The scale bar is 30 ␮m.

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Table 1 Distribution and number of urocortin and CRF immunoreactive cells in the central nervous system of Lumbricus terrestris Ganglia

Location

Number of urocortin immunopositive cells

Number of CRF immunopositive cells

Medial Dorsomedial Dorsolateral Lateral Central

4–12 0 1 0–2 1–3 2–6

31–52 1–4 6–10 12–21 11–15 1–2

Medial Ventromedial Ventrolateral Lateral

30–75 6–10 12–30 8–25 4–10

10–20 1–2 3–6 4–7 4–7

Medial Ventromedial Ventrolateral Lateral

57–88 9–14 18–22 20–28 10–24

8–24 1–4 2–6 3–10 2–4

Cerebral

Subesophageal

Ventral

atogastric ganglia. The fibers of the connectives displayed strong immunoreactivity to Ucn (Fig. 2E) and CRF. 3.2. Subesophageal ganglion The subesophageal ganglion is formed by the fusion of the three cranial ganglia of the ventral chain. The rostral part communicates predominantly with the cerebral ganglion, while the caudal part is similar in structure to a ventral cord ganglion. In contrast to the cerebral ganglion, most neurons of the subesophageal ganglion are located in the ventral part, surrounding the central neuropil [6]. The cells are distributed into medial, ventromedial, ventrolateral and lateral cell groups. The number of neurons is approximately 1500 [2]. Ucn- and CRF-like ir cells were found in all cell groups of the subesophageal ganglion, in ventromedial, ventrolateral and lateral positions (Figs. 1, 2C and D, 3B and C). The number of Ucn-like ir neurons was higher, representing 2–5% of the total cell number (Table 1). The CRF-like immunopositive cells were less than in the cerebral ganglion, representing only 0.7–1.4% of the total cell number in the subesophageal ganglion (Table 1). The morphology of the immunoreactive neurons was similar. The ventromedial and ventrolateral cells were pyriform in shape, measuring 15–20 ␮m, with their axons running towards the central neuropil. The lateral cells were smaller (10–15 ␮m), oval or round in shape, and their axons were only rarely visible. The central neuropil contained scattered immunopositive fibers (Fig. 2D).

per ganglion [2]. The morphology and location of the Ucnand CRF-like ir cells was found to be similar to that in the subesophageal ganglion (Figs. 1, 2F and G, 3D, E, G, and H). They represented 4.1–6.3 and 0.6–1.7% of the total cell number, respectively (Table 1). 3.4. Peripheral nervous system Ucn-like immunoreactivity was found only in the enteric system of the earthworm. The stomatogastric nervous system of invertebrates consists of peripheral ganglia and nerve fibers, and is comparable in function to the autonomic nervous system of vertebrates [16]. Nerves emerging from the stomatogastric and ventral cord ganglia form the enteric plexus that corresponds to the myenteric plexus of vertebrates [16]. A smaller percentage of the fibers forming the enteric plexus are of intrinsic origin, from the solitary nerve cells found in the mucosa of the digestive tract [6]. Ucn-like immunoreactivity was found only in the cranial part of the pharyngeal plexus. Several Ucn-like ir cells were seen in the pharyngeal wall as well as a rich immunoreactive fiber network was found in the subepithelial and submucosal locations in the pharyngeal wall (Fig. 4A and B). No immunopositive cells were seen in the stomatogastric ganglia. The rest of the enteric nervous system did not display any immunoreactivity. No immunopositive cells or fibers were observed with CRF antiserum.

4. Discussion 3.3. Ventral cord ganglia The subesophageal ganglion continues in the ventral ganglionic chain, which runs along the length of the earthworm. Similarly to the subesophageal ganglion, the neurons are situated in the ventral part, approximating 1400 cells

In the present account, the distribution of Ucn- and CRF-like immunoreactivities was studied in the central nervous system of the earthworm, and for the first time, morphological evidence for the existence of Ucn- and CRF-like compounds in L. terrestris is provided. Members of the

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CRF neuropeptide family represent distinct neuropeptides in various lower vertebrates and their structures are different from that of Ucn [9,25]. In addition, the distinct distribution of CRF and Ucn in the earthworm CNS (Fig. 1) suggests that these neuropeptides are involved in distinct biological functions in the earthworm central nervous system. Our results conform to the findings of Remy et al. [32] that CRF-like immunoreactivity is present in the annelids. The morphology of CRF-like ir neurons described in our present study was also found to be similar to those described by Remy et al. [32]. However, the quantitative distribution of CRF-like immunoreactivity revealed some differences, since we found CRF-like ir perikarya to be most abundant in the cerebral ganglion, whereas Remy et al. [32] found most of the CRF-like ir neurons confined to the ventral cord ganglia. This discrepancy can be explained by the different species and methodologies used in these studies. Following the discovery of the peptide family called urotensins, several structurally similar peptides, such as urotensin I (U-I), sauvagine and CRF were isolated [9]. Moreover, the detailed characterization and receptor binding studies of urotensins [35] raised the possibility that additional members of this peptide family are present in the brain. Indeed, molecular screening studies, using a carp U-I cDNA probe, a unique cDNA was cloned, which encoded a precursor and a putative peptide named Ucn, referring to its 63% sequence identity to U-I and 45% sequence identity to CRF [36]. Recently, two new members of the CRF neuropeptide family, named urocortin II and urocortin III (stresscopin) have been isolated [9,24]. The amino acid sequence of Ucn is only 20% similar to that of Ucn II or III [24]. Although the Ucn antibody used in this study was not tested for cross-reactivity with these newly identified members, based on phylogenetic and structural differences [24], it is unlikely that anti-Ucn serum detects mammalian Ucn II or III to any appreciable extent (personal communication to Prof. W.W. Vale). However, we cannot rule out the possibility that annelid Ucn II or III (if they exist) might have a few changes in phylogeny that would permit cross-reactivity. Further studies would be needed using specific antibodies against Ucn II or III to address this problem. The sequence identity in the structure of human, rat and frog CRF indicates that this compound is highly preserved in all vertebrate species [25]. Moreover, in view of phylogeny, it is intriguing that a duplication of an early gene of CRF neuropeptide family has been postulated, resulting in a divided phylogenetic tree [24]. Ucn II and III represent a separate branch, whereas human/ovine CRF, frog sauvagine, pufferfish urotensin and human/mouse Ucn are all members of the same branch of this phylogenetic tree [24]. It has also been suggested that this gene duplication occurred early in phylogeny [24]. However, the species where this ancestor gene divided remains at issue. The results of Morley et al. [28] suggest that distinct teleost U-I and CRF genes, arising from gene duplication, may have occurred early in vertebrate lineage. Furthermore, the demonstration that several

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diuretic peptides from insects possess structural similarity to U-I and CRF indicates that both peptides may have arisen prior to the appearance of chordates [7]. Our present results on the differential distribution of CRF and Ucn in the earthworm suggest that both peptides appeared early in species development and are well conserved in phylogeny. CRF exerts a wide range of actions in the central nervous system of vertebrates [11]. Among others, it increases arousal and produces behavioral manifestations of the state of stress [19]. At present, less is known about the functions of Ucn. Along with other members of the CRF peptide family, Ucn elicits many of the same biological actions as CRF in response to stress and plays an important role in modifying the efferent components of endocrine, autonomic and immune responses [19,35,39]. The cellular morphology of the central nervous system of Oligochaeta has been well documented by a number of investigators [6,15], and has been shown to integrate sensory processes, regeneration, feeding, and motor activity. However, functionally identified nerve cells are not as well known as in other invertebrates. The functional identification of the Ucn and CRF immunostained cells is therefore difficult, and is further hindered by the faint or lack of staining of neurites. However, the high number of Ucn- and CRF-like ir cells and functional reports on CRF-related peptides in other invertebrates allow some general remarks to be made. Stress phenomena have been shown to occur also in invertebrates, and the influence on the immune response appears to be mediated by neuropeptides similar to those involved in the vertebrate stress response [29,30]. The earthworm also possesses various reflex mechanisms in response to stress, which involves central integration of sensory inputs, motor coordination and immune responses [6,8]. An ancestral type of stress response is mediated by CRF and its fragments in molluscan hemocytes [13,29,30], which implies an ancient neuroimmunoregulatory role of CRF and related peptides. Also, CRF has been suggested to be a neurotransmitter in antennal receptors in insects [37]. In the cerebral ganglion of the earthworm, our present data revealed numerous small CRF-like ir perikarya, which most probably belong to the group of nerve cells designated as small interneurons [6]. Similar to the molluscan hemocytes, the earthworm possesses the so-called celomocytes, which are the main effector cells of the immune system [8]. Antigen-binding properties of the celomocytes have been thoroughly studied, but no data are available about the neurohormonal regulation of these cells [4,8]. Similarly to other invertebrates, the CRF- and Ucn-like ir neurons in the earthworm may be involved in integrating peripheral sensory information, central pathways and neuroimmune responses in response to external stimuli. This is further implied by the presence of ACTH in the earthworm central nervous system [18], for ACTH being one of the effectors of CRF-related actions not only in vertebrates but also in invertebrates [29]. Although the presence of Ucn has not yet been reported in invertebrates, several other members of the CRF peptide

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family have been isolated and described in insects and molluscs [7,14,17]. Some of these peptides seem to be specific to invertebrates, and play an important role in the neuroendocrine regulation of fluid secretion [7,14]. Neuropeptides, such as oxytocin and vasopressin also participate in the fluid homeostasis of the earthworm and the involvement of the central nervous system has been reported in the control of dehydration [12,40], and yet the regulation of earthworm excretory system has not been studied in such details as in other invertebrates. However, based on the widespread occurrence of CRF- and Ucn-like immunoreactivities in the nervous system of L. terrestris, it cannot be excluded that similarly to other invertebrates, CRF and Ucn participate in the regulation of fluid homeostasis in Annelida. Ucn is also present in the rat enteric nervous system, and similarly to CRF, Ucn reduces food intake and delays gastric emptying in mammals [3,22]. CRF-like ir cells and fibers have been demonstrated also in the mid- and hindgut of insects [33]. In the present study, Ucn-like ir structures were observed in the pharyngeal wall of the earthworm. These fibers most probably originate from the cerebral and subesophageal ganglia, since no immunoreactive cells were found in the stomatogastric ganglia. This finding suggests that Ucn may be involved in the central regulation of the activity of the pharyngeal wall, thereby regulating/modulating the feeding activity of the earthworm. In summary, this study, for the first time revealed that Ucn and CRF-like compounds are present in various neuronal structures of the earthworm. Furthermore, these findings are in concert with the results of the literature that the distributions of these peptides do not overlap in general, suggesting distinct biological significance for CRF and Ucn. Based on the data presented in this study and those of the literature, it can be postulated that Ucn and CRF neuronal structures are well preserved during phylogeny and they may represent two parallel neuronal circuits involved in the regulation/modulation of several physiological and endocrine functions also in the annelid L. terrestris.

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