Endomorphin-1 (Tyr-Pro-Trp-Phe-NH2, EM1) and endomorphin-2 (Tyr-Pro-Phe-Phe-NH2, EM2) are the endogenous ligands with high affinity and selectivity for the μ-opioid receptor (MOR) (Thompson et al.,1993; Zadina et al.,1997,1999; McConalogue et al.,1999; Wang et al.,2003). EM1- and EM2-immunoreactive (EM1- and EM2-IR) neuronal cell bodies are localized principally in both hypothalamus and nucleus tractus solitarii (NTS) of the central nervous system (CNS). However, EM1- and EM2-immunopositive axons and axon terminals are distributed in many regions of the CNS, including the parabrachial nuclei (PBN) (Martin-Schild et al.,1999; Pierce and Wessendorf,2000; Greenwell et al.,2002). In previous studies, we have demonstrated that EM1- and EM2-IR neurons possess reciprocal connections between the hypothalamus and NTS (Hui et al.,2006), and that EM1- and EM2-IR hypothalamic neurons project to the PBN (Chen et al.,2002). The hypothalamus, PBN, and NTS play important roles both in the visceral and somatic functions (Hylden et al.,1989; Tache et al.,1990; Slugg and Light,1994; Tseng et al.,2000; Boscan and Paton,2001; Boscan et al.,2002; Simons et al.,2003; Glatzer and Smith,2005; Zhang and Ennis,2007). However, it is poorly understand whether the endomorphinergic projection fibers from the NTS might terminate in the PBN. Therefore, the present study utilized EM1 or EM2 immunofluorescent stainings, in combination with fluorescent retrograde or anterograde tracing, to examine whether EM1- and EM2-IR neurons in the NTS sent their axons to the PBN in the rat.
Endomorphin-1 (EM1) and endomorphin-2 (EM2) are the selective endogenous ligands for the μ-opioid receptor (MOR). Since EMs-expressing neuronal cell bodies or axonal components have been observed, respectively, in the nucleus tractus solitarii or the parabrachial nuclei, we examined if EMs-expressing neurons in the NTS of the rat might send their axons to the PBN. Immunofluorescent stainings for EM1 or EM2 were combined with retrograde or anterograde tract-tracing method. After injecting tetramethyl rhodamine dextran-amine (TMR) into the parabrachial nuclei of rats, some EM1- or EM2-immunoreactive neurons in the nucleus tractus solitarii were labeled retrogradely with TMR. The majority of the EM1/TMR and EM2/TMR double-labeled neurons were observed in the medial, commissural, and dorsolateral subnuclei of the nucleus tractus solitarii. Following injection of biotinylated dextran amine (BDA) into the medial, commissural, or dorsolateral subnuclei of the nucleus tractus solitarii, EM1- or EM2-immunopositive axons and axon terminals were anterogradely labeled with BDA mainly in the lateral parabrachial nucleus. The present results have indicated that endomorphinergic neurons in the nucleus tractus solitarii project to the parabrachial nuclei. This suggests that EMs released from NTS-PBN projection fibers may bind to MOR on the PBN neurons to be implicated in processing of visceral information within the parabrachial nuclei. Anat Rec, 292:488–497, 2009. © 2009 Wiley-Liss, Inc.
MATERIALS AND METHODS
A total of 22 male Sprague-Dawley rats, weighing 250–300 g, were used for the present study. All procedures were approved by the Committee of Animal Use for Research and Education of the Fourth Military Medical University (Xi'an, People's Republic of China). Ten rats were used for retrograde tracing, and the remaining rats were used for anterograde tracing.
In each rat anesthetized by intraperitoneal injection (i.p.) of sodium pentobarbital (40 mg/kg body weight), a single injection of 0.2–0.3 μL of 10% (w/v) tetramethyl rhodamine dextran-amine (TMR, molecular weight 3000; Molecular Probes, Eugene, Oregon) was made stereotaxically into the right PBN according to the atlas of the rat brain (Paxinos and Watson,1999). After 5–7 days, all rats were anesthetized with an overdose of sodium pentobarbital (100 mg/kg body weight, i.p.) and were perfused transcardially with 100 mL of 0.9% saline, followed by 500 mL of 0.1 M phosphate buffer (PB, pH 7.4) containing 4% (w/v) paraformaldehyde and 0.2% (w/v) picric acid. The brains were immediately removed and placed into the same fresh fixative for 2 hr at 4°C. Subsequently, the brains were placed into 30% (w/v) sucrose in 0.1 M PB (pH 7.4) overnight at 4°C, and then cut serially into 30-μm-thick frontal sections using a freezing microtome (Kryostat 1720; Leitz, Mannheim, Germany). The sections were serially collected into four dishes containing 0.01 M phosphate-buffered saline (PBS, pH 7.4) as four sets of every fourth serial section (each dish had about 15–20 serial sections). All sections in all dishes were washed with 0.01 M PBS. The sections in the first dish were mounted onto gelatin-coated glass slides without cover-slips and used to examine the TMR injection site in the PBN and the distribution of retrogradely TMR-labeled neurons in the brainstem, especially in the NTS. Flowing observation, these sections were processed for Nissl staining.
The sections in the second and third dishes were used for EM1- and EM2-immunoflurescent stainings, respectively. The sections were sequentially incubated at 4°C with (1) rabbit antiserum against EM1 (AB5102, 1:100 dilution; Chemicon, Temecula, CA) or rabbit antiserum against EM2 (AB5104, 1:200 dilution; Chemicon) in 0.0l M PBS containing 5% (v/v) normal donkey serum (NDS), 0.3% (v/v) Triton X-100, 0.05% (w/v) NaN3, and 0.25% (w/v) lambda-carrageenan (PBS-NDS, pH 7.4) for 72 hr; (2) biotinylated goat anti-rabbit IgG (1:200 dilution; Chemicon) in PBS-NDS for 4 hr; (3) fluorescein isothiocyanate (FITC) labeled avidin D (1:1000 dilution; Vector, Burlingame, CA) in 0.01 M PBS containing 0.3% (v/v) Triton X-100 (PBS-X) for 2 hr. Between each step, the sections were washed thoroughly with PBS-X. The immunofluorescent-stained sections were mounted onto clean glass slides, air dried, cover-slipped with 0.01 M PBS containing 50% (v/v) glycerin and 2.5% (w/v) triethylene diamine (antifading reagent). The sections were analyzed using laser scanning confocal microscope (FV1000; Olympus, Tokyo, Japan) with the appropriate monochromatic lasers for TMR (excitation 570 nm; emission 600 nm) and FITC (excitation 490 nm; emission 520 nm), respectively.
The sections in the fourth dish served as controls. In the control experiment, the primary antibodies were omitted or replaced with normal rabbit serum. The remaining procedures were the same as for the sections in the second and third dishes. No immunopositive products were detected.
Twelve rats were used for anterograde tracing. In each rat anesthetized with sodium pentobarbital (40 mg/kg body weight, i.p.), a single injection of 0.2–0.3 μL of 10% (w/v) biotinylated dextran amine (BDA, molecular weight 10,000; Molecular Probes) was stereotaxically made into the right medial (N = 4), commissural (N = 4), or dorsolateral subnucleus (N = 4) of the NTS according to the atlas of the rat brain (Paxinos and Watson,1999). After 5–7 days, all rats were identically to the retrograde tracing animals. Subsequently, the brains were serially cut into 30-μm-thick frontal sections. The sections were serially collected into three dishes containing 0.01 M PBS (pH 7.4). The sections in the first and second dishes were used for EM1- and EM2-immunofluorescent stainings, respectively. The sections were sequentially incubated at 4°C with (1) rabbit antiserum against EM1 (AB5102, 1:100 dilution; Chemicon) or rabbit antiserum against EM2 (AB5104, 1:200 dilution; Chemicon) in PBS-NDS (pH 7.4) for 72 hr; (2) a mixture of FITC conjugated donkey anti-rabbit IgG (1:200 dilution; Chemicon) and Cy3-conjugated streptavidin (1:1000 dilution; Jackson ImmunoResearch Lab, PA) in 0.01 M PBS-X for 4 hr. Between each step, the sections were washed with PBS-X. The sections were then mounted onto clean glass slides, air dried, cover-slipped with 0.01 M PBS containing 50% (v/v) glycerin and 2.5% (w/v) triethylene diamine. Then the sections were analyzed using laser scanning confocal microscope (FV1000; Olympus) with the appropriate monochromatic lasers for Cy3 (excitation 552 nm; emission 565 nm) and FITC (excitation 490 nm; emission 520 nm), respectively.
The sections in the third dish were used for empty or replace control tests. No immunopositive products were detected.
A total of 7/10 rats presented with successful TMR injections into the right PBN, including the medial parabrachial nucleus (MPB) and lateral parabrachial nucleus (LPB), without involving the mesencephalic trigeminal nucleus adjacent to the PBN (Fig. 1). After injecting TMR into the right PBN, retrogradely TMR-labeled neurons were mainly distributed in the ipsilateral spinal trigeminal nucleus, NTS, gracile nucleus, cuneate nucleus, and medullary reticular nuclei of the brainstem. Within the NTS, TMR-labeled neurons were mainly found at the level of the area postrema (AP), in particular in the medial (mNTS), commissural (comNTS), and dorsolateral (dlNTS) subnuclei of the NTS with predominance ipsilateral to the TMR injection (Figs. 2C, 3C, and 4). A few retrogradely TMR-labeled neuronal cell bodies were also observed in the gelatinous and ventral subnuclei of the right NTS. In the NTS on the side contralateral to the TMR injection into the PBN, only a few neuronal cell bodies were labeled retrogradely with TMR. These TMR-labeled neurons varied from small to intermediate in size and round, triangular, or elliptic in shape (Figs. 2C and 3C). The diameters of these TMR-labeled neurons were about 10–35 μm.
The NTS also contained EM1- or EM2-immunopositive neuronal cell bodies (Figs. 2A,B, 3A,B, and 4). EM1-IR neurons were distributed in the mNTS, comNTS, dlNTS, and gelatinous subnuclei of the NTS at the level of the AP, while EM2-IR neurons were mainly observed in the mNTS and dlNTS at the same level and just rostral to the level of the AP (Figs. 2A,B and 3A,B). Both EM1- and EM2-IR neurons presented with small and intermediate neuronal somata (with diameter in 10–25 μm) and exhibited round, triangular, or elliptical shapes (Figs. 2A,B and 3A,B). These results were in good accordance with reports from previous studies (Martin-Schild et al.,1999; Pierce et al.,2000; Hui et al.,2006).
After the TMR injection into the PBN, some of EM- and EM2-immunopositive neuronal cell bodies in the NTS on the side ipsilateral to the TMR injection were labeled retrogradely with TMR (Figs. 2D and 3D). These EM1/TMR or EM2/TMR double-labeled neurons shared similar morphological characteristics with EM1- or EM2-IR neurons and were primarily observed in the mNTS and dlNTS ipsilateral to the TMR injection site in the PBN. A few EM1/TMR double-labeled neurons were also found in the comNTS and gelatinous subnuclei of the NTS. There were no EM1/TMR or EM2/TMR double-labeled neurons in the left NTS (Fig. 4). In the NTS on the side ipsilateral to the TMR injection into the PBN, neuronal cell bodies which were dually labeled with EM1/TMR or EM2/TMR constituted 9.4% or 5.8% of the total population of TMR-labeled neuronal cell bodies in the NTS, respectively, and 9.1% or 6.5% of the total population of EM1- or EM2-immunopositive neuronal cell bodies in the NTS, respectively (Table 1); EM1/TMR-labeled neuronal cell bodies were more numerous than EM2/TMR-labeled ones (Table 1).
|Rat||TMR-labeled neurons||EM1-IR neurons||EM1/TMR double-labeled neurons (%1; %2)||TMR-labeled neurons||EM2-IR neurons||EM2/TMR double-labeled neurons (%1; %2)|
|R1||78||86||9 (11.5; 10.5)||89||73||6 (6.7; 8.2)|
|R3||87||94||7 (8.0; 7.4)||78||76||4 (5.1; 5.3)|
|R4||80||79||8 (10.0; 10.1)||96||82||3 (3.1; 3.7)|
|R5||95||103||10 (10.5; 9.7)||103||83||5 (4.9; 5.4)|
|R7||69||72||5 (7.2; 6.9)||80||69||4 (5.0; 5.8)|
|R8||74||70||8 (10.8; 11.4)||82||73||7 (8.5; 9.6)|
|R9||83||81||6 (7.2; 7.4)||77||81||6 (7.8; 7.4)|
|Mean||80.9||83.6||7.6 (9.4; 9.1)||86.4||76.7||5.0 (5.8; 6.5)|
The BDA injection was made into each of the NTS subnuclei where neuronal cell bodies dually labeled with EM1/TMR or EM2/TMR had been detected frequently: After the BDA injection into the mNTS (Fig. 5), comNTS, or dlNTS, BDA-labeled axons and axon terminals were seen in the PBN regions; mainly in the LPB, MPB, and Kölliker-Fuse nucleus (KF). The BDA-labeled axonal components were distributed more densely in the LPB than in the MPB, and were also scattered in the KF (Figs. 6–8). Within the PBN, almost all of EM1- or EM2-immunopositive axonal components that were anterogradely labeled with BDA were distributed in the LPB, while only a small number of them were also seen in the MPB and KF (Figs. 6–8).
The present results have indicated that EMs-expressing neurons in the NTS send their axons to the PBN in the rat. The PBN receives projection fibers not only from the NTS but also from the superficial laminae of the medullary and spinal dorsal horns, and is implicated in the transmission and modulation of special visceral (gustatory) information and the general visceral and somatic information including nociceptive information (Li et al.,1990; Chiang et al.,1994; Ding et al.,1995,1996a; Li and Li,2000; Glatzer and Smith,2005). Among the different subnuclei of the PBN, the LPB is a relay for visceral and autonomic afferents (Herbert et al.,1990), the MPB is a relay in the gustatory pathways (Hansell and Frank,1991), and the KF is implicated in control of respiratory functions (Chamberlin and Saper,1992; Dick et al.,1994).
In the CNS, it has been confirmed that EMergic neuronal cell bodies are localized in the hypothalamus and NTS, while EMs-IR fibers and terminals are distributed much more widely (Martin-Schild et al.,1999; Pierce and Wessendorf,2000). Our previous study demonstrated that hypothalamic EMs-IR neurons topographically send projections to the PBN (Chen et al.,2002). In the present study, we observed that EM1- and EM2-IR neurons in the mNTS, comNTS, and dlNTS send projections to the PBN, mainly to the LPB. Based on the results from our previous study (Chen et al.,2002) and the present results, it is reasonable to conclude that EMergic fibers and terminals in PBN originate from both the hypothalamus and the NTS. Since it has been well known that EMs have analgesic effect in the CNS (Stone et al.,1997; Zadina et al.,1997; Narita et al.,1998; Tseng et al.,2000), the PBN that receives endomorphinergic fibers from the hypothalamus and NTS is considered to play important roles in the processing of nociceptive information from both somatic and visceral regions. EMs released from axon terminals of endomorphinergic fibers within the PBN may bind to MOR expressed on PBN neurons, which are known to be distributed extensively in the PBN (Mansour et al.,1995; Ding et al.,1996b; Chamberlin et al.,1999; Neal et al.,1999).
In summary, by using fluorescent retrograde and anterograde tracing methods combined with immunofluorescent stainings of EM1 or EM2, we demonstrated that some EM1- and EM2-IR neurons in the NTS sent ascending EMergic fibers to the PBN, in particular to the LPB. These EMergic ascending fibers from the NTS to the PBN might affect the functions of the PBN by releasing EM1 and EM2, which bind to the MOR expressed on the PBN neurons.