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Localization of neuropeptide Y Y1 receptor-like immunoreactivity in catecholaminergic neurons of the rat medulla oblongata
Pergamon
Neuroscience Vol. 73, No. 2, pp. 519 530, 1996 Copyright © 1996 IBRO. Published by ElsevierScience Ltd Printed in Great Britain S0306-4522(96)00072-3 0306-4522/96 $15.00 + 0.00
LOCALIZATION OF NEUROPEPTIDE Y Y1 RECEPTOR-LIKE IMMUNOREACTIVITY IN CATECHOLAMINERGIC NEURONS OF THE RAT MEDULLA OBLONGATA S.-N. Y A N G , * t B. B U N N E M A N N , : ~ A. C I N T R A * and K. F U X E * *Division of Cellular and Molecular Neurochemistry, Department of Neuroscience, Karolinska Institutet, Doktorsringen 12, S-171 77 Stockholm, Sweden ++Department of Pharmacology, Glaxo Research Laboratory, Verona, Italy Abstract--Neuropeptide Y receptors in the medulla oblongata participate in central cardiovascular control. The neuropeptide Y~ receptor subtype gene and amino acid sequence have been identified by molecular cloning studies. In this study, a C-terminal peptide representing amino acids 355-382 of the neuropeptide Yt receptor was synthesized and cross-linked to thyroglobulin to produce an antibody against a partial sequence of the neuropeptide Y~ receptor, used to localize neuropeptide Y~ receptor-like immunoreactivity in the catecholaminergic neurons of the medulla oblongata. The double colour immunofluorescence technique with a polyclonal antibody against the neuropeptide Yt receptor and a monoclonal antibody against tyrosine hydroxylase revealed that in the rat medulla oblongata, a weak (the C3 cell group) to moderately intense (the A 1, A2, C 1 and C2 cell groups), diffuse cytoplasmic neuropeptide Y~ receptor-like immunoreactivity was distributed primarily in the noradrenergic and adrenergic cell bodies and occasionally seen in the noradrenergic and adrenergic cell processes. Almost all tyrosine hydroxylase-like immunoreactive cell bodies in the AI, A2, CI, C2 and C3 cell groups showed neuropeptide Yt receptor-like immunoreactivity. The neuropeptide YI receptor-like immunoreactivity in the A2 cell group was somewhat stronger. The present findings show localization of specific neuropeptide Yt receptor-like immunoreactivity in the vast majority of the noradrenergic and adrenergic cell bodies of the A1, A2, CI, C2 and C3 cell groups, which are putative cardiovascular regions. The results support the view that neuropeptide Y~ receptors in the medulla oblongata are involved in central cardiovascular control and may coexist with another important receptor, the %A-adrenoceptor, also involved in central cardiovascular regulation, since the cqA-adrenoceptor-like immunoreactivity has been shown to exist in almost all noradrenergic and adrenergic cell bodies in the brainstem. In conclusion, centrally administered neuropeptide Y may act in part via neuropeptide Y~ receptors located on the soma and dendrites of noradrenergic and adrenergic neurons, where it may interact with c~2-adrenoceptors at least in the noradrenergic A2 neurons. This noradrenaline system may be involved in at least part of the vasodepressor actions of neuropeptide Y, noradrenaline and adrenaline in the nucleus tractus solitarii in view of the present findings. Copyright .© 1996 IBRO. Published by Elsevier Science Ltd. Key words: medulla oblongata, adrenergic, noradrenergic, peptide receptor, immunocytochemistry, tyrosine hydroxylase.
effects. 24'49'66 However, only the N P Y YI receptor gene has been cloned) 6"3s'41'56 It has been shown that the N P Y YI receptor has seven transmembrane domains, three extracellular and three intracellular loops like other members of the G-protein-coupled superfamily of receptors. 16'18'19'33'4°'49'63 In situ hybridization studies have demonstrated that N P Y Yj receptor m R N A s are strongly expressed in peripheral and central neurons. 37'42'43'5°'69The adrenergic and noradrenergic nuclei in the medulla oblongata responsible for regulating cardiovascular activity 3'8'9"H'21 contain a high density of N P Y Y~ receptor binding sites. 2'25'44,46 However, autoradiographical evidence suffers from the lack of cellular resolution and thus does not establish the existence of binding sites on neurons and/or on glia,
Neuropeptide Y (NPY) is a putative regulatory peptide in the brain and participates in the regulation of several physiological functions including the autonomic activity, especially cardiovascular control, food intake, circadian rhythm, m e m o r y and neuroendocrine regulation. 4A3'14'52'61'62 These physiological actions of N P Y depend on the activation of N P Y receptors. Five N P Y receptor subtypes, YI, Y2, Y3, " F e e d i n g " and " P Y Y preferring", have been identified according to their pharmacological properties and physiological tTo whom correspondence should be addressed. -LI, -like immunoreactivity or immunoreactive; NPY, neuropeptide Y; PBS, phosphatebuffered saline; TH, tyrosine hydroxylase.
Abbreviations:
519
520
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and does not identify the neurochemical features of cells with N P Y YI receptor binding sites. N P Y Y~ receptors do not always achieve their physiological effects independently. They often do so interacting with other receptors such as ~2-adrenoceptors and angiotensin II receptors involving G-protein-dependent intramembrane recepto~receptor interactions. 1"2°'22'23"3°'36"65"67'68 The findings from this laboratory indicate that activation of N P Y YI receptors in the nucleus tractus solitarii can significantly decrease the affinity of high-affinity c~2-adrenoceptor binding sites and substantially counteract the ~2-adrenoceptor-mediated vasodepressor and bradycardic responses in the nucleus tractus solitarii. 6768 On the basis of these findings it can be postulated that the N P Y Y~ receptor may be localized together with e2-adrenoceptors in neurons of the nucleus tractus solitarii. More recent evidence indicates that e2Aadrenergic receptor-like immunoreactivity (-LI) is present in almost all noradrenergic and adrenergic cells of the brainstem. 29"53-57 In the present study an antibody against a C-terminal portion of the N P Y Y~ receptor (see Ref. 69) was employed to study the possible coexistence of NPY Y~ receptor-LI and tyrosine hydroxylase-LI (TH-LI) in the A1, A2, CI, C2 and C3 cell groups in the medulla oblongata in order to identify the neurochemical features of neurons with N P Y Yj receptor-LI in the medulla oblongata. Such a study will also indirectly give indications on the co-localization of N P Y Y~ receptors and ~2-adrenoceptors. Therefore there exists a potential to obtain an important morphological substrate for NPY Y~/e2 receptor~receptor interactions in the nucleus tractus solitarii known to be involved in cardiovascular c o n t r o l . 67,68
EXPERIMENTAL PROCEDURES
Animals
Male specific pathogen-free Sprague Dawley rats (180 200g body weight; B & K Universal, Stockholm, Sweden) were used. They were given food pellets and water ad libitum and kept under standardized temperature, humidity and lighting conditions (lights on at 8.00 a.m. and off at 8.00 p.m.).
Leitz cryostat at the analysed bregma level according to the atlas of Paxinos and Watson? 4 The sections were collected in the test tube and processed by the two colour immunofluorescence technique with the free-floating method. The sections were incubated for 48 h with a mixture of the rabbit antibody against the NPY Y~ receptor (1:5000 in 0.1 M PBS/0.5% Triton X-100) and the monoclonal mouse antibody against rat TH (1:800 in 0.1 M PBS/0.5% Triton X-100; INCSTAR Co. Minnesota, U.S.A.). After washing 3 x l0 min in 0.1 M PBS, the sections were incubated for 4 h at room temperature with a mixture of fluoresceine isothiocyanate-conjugated donkey anti-mouse immunoglobulins (1:40 in 0.1 M PBS/0.5% Triton X-100, Jackson, West Grove, PA) and CY3VM-conjugated AffiniPure goat antirabbit IgG (1 : 100 in 0.1 M PBS/0.5% Triton X-100, Jackson, West Grove, PA). The sections were then rinsed three times for 10min in 0.1 M PBS and finally mounted in an antifading medium and examined in a Nikon Microphot-FX epifluorescence microscope equipped with the appropriate excitation and barrier filter combinations. The specificity of the antibody was tested by absorption with the synthetic C-terminal peptide (amino acids 355 382) in a concentration of 20/*g/ml diluted antibody. One representative section from each brain at the same bregma level was selected and examined in a Nikon Microphot-FX microscope with a x 10 or x20 objective depending upon the area. The number of NPY Y~ receptorLI and TH-Li cells was counted within the AI, A2, CI, C2 and C3 celI groups, respectively. The cumulated counts from five rats were reported.
RESULTS
A moderately intense, diffuse cytoplasmic N P Y Yl receptor-LI was observed in the noradrenergic AI and A2 cell groups as well as in the adrenergic C1 and C2 cell groups, being somewhat stronger in the A2 cell group. A weak, diffuse cytoplasmic NPY Yl receptor-LI was present in the adrenergic C3 cell group. The appearance of the immunoreactivity was fully blocked by preabsorption of the antibody with the synthetic C-terminal peptide representing a partial sequence of the N P Y YI receptor. As seen in Fig. 1A, a diffuse cytoplasmic NPY Y~ receptor-LI existed in the noradrenergic A2 cell group area, but no NPY Yl receptor-LI was observed in the same area of an adjacent section preabsorbed with the synthetic C-terminal peptide (20/~g/ml diluted antibody; Fig. 1B). The same results were observed in the A I , C1, C2 and C3 cell groups.
Preparation (~["antibodies
A C-terminal peptide representing amino acids 355 382 of the NPY Y~ receptor was synthesized, cross-linked to thyroglobulin and injected into two rabbits. Sera were collected after two boosters and then purified. 69 Immunoeytochemist O,
Five rats were anaesthetized with sodium pentobarbital (60mg/kg, i.p.). The transcardiac perfusion was then performed by using a fixative consisting of 2% paraformaldehyde and 14% v/v saturated picric acid in 0.1 M sodium phosphate buffer (pH 7.3). The brain was rapidly dissected and postfixed by immersion in the fixative solution for 90min and subsequently washed twice in 0. I M phosphate-buffered saline (PBS) for 10min. The brains were then placed in 0.1 PBS containing 20% sucrose for 24h. Coronal sections (50/~m thick) were cut with a
A 1 cell group
A prominent cytoplasmic T H - L I and moderately intense, diffuse cytoplasmic N P Y Y~ receptor-LI was observed in the noradrenergic A1 cell group area of the caudal medulla oblongata, located in the ventrolateral part of the medullary reticular formation. As seen in Fig. 2, the N P Y Y~ receptor-Ll was exclusively present in T H - L I cell bodies and in dendritic processes. Ninety-six per cent of the T H - L I neurons were positive for N P Y Y~ receptor-LI and specific N P Y Y~ receptor-LI profiles were not present in the TH-negative cell bodies and processes within the noradrenergic A1 cell group area (Fig. 2, Table I).
NPY YL receptor-LI in catecholaminergic neurons A 2 cell group
As shown in Fig. 3, a cytoplasmic TH-LI of strong intensity and diffuse cytoplasmic NPY Y1 receptor-LI of moderate intensity appeared in the noradrenergic A2 cell group area of the medial subnucleus of the nucleus tractus solitarii. The TH-LI was also seen in the nerve terminals. A few TH-LI cell body profiles were scattered in the dorsal motor nucleus of the vagus, the intermediate subnucleus of the nucleus tractus solitarii and the commissural nucleus. The NPY Y~ receptor-Ll was almost exclusively present in TH-LI cell bodies. A few TH-LI dendrites were also NPY Y~ receptor positive. The vast majority of TH-LI cells (98%) were double-labelled (Table 1) and specific NPY Y~ receptor-LI profiles
A
Gr
521
were not seen in the TH-negative cell bodies and processes within the noradrenergic A2 cell group area (Fig. 3). C I cell group
Moderately intense, diffuse cytoplasmic NPY Yt receptor-LI and strong cytoplasmic TH-LI cell body profiles appeared in the adrenergic C1 cell group, mainly found in the rostral ventrolateral part of the medullary reticular formation. The NPY Yj receptorLI was exclusively found in TH-LI cell bodies and in dendritic processes. Ninety-nine per cent of the THLI neurons were double-labelled with the NPY Y1 receptor antibody (Fig. 4, Table 1). Moreover, specific NPY Yl receptor-LI profiles were not
NpY Y1-L| AP
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Fig. I. A substantial number of cell body profiles with diffuse cytoplasmic NPY Y1 receptor-LI (arrows) of moderate intensity exists in the noradrenergic A2 cell group area (A), but no NPY Y~ receptor-LI (B) is seen in the same area of the adjacent section, where the NPY Yt receptor antibody has been preabsorbed with the synthetic peptide, representing a partial sequence of the NPY Yt receptor. The indirect immunofluorescencetechnique has been used. AP, area postrema; Gr, gracile nucleus; NTS, the nucleus tractus solitarii; asterisk, central canal. Bregma level -14.0 mm. Bar = 100 #m.
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In this adrenergic cell group, cell body profiles with diffuse cytoplasmic NPY Y~ receptor-LI of moderate intensity and with cytoplasmic TH-LI of strong intensity were found on the dorsal border of the rostral nucleus tractus solitarii. A dense network of TH-LI terminals appeared in the medial pole of the nucleus tractus solitarii. The NPY Y~ receptor-LI was almost exclusively observed in TH-LI cell bodies. The percentage co-localization of TH-LI and NPY Y1 receptor-LI was 93% in relation to TH-LI neurons (Fig. 5, Table 1). Specific NPY YI receptor-LI profiles did not appear in the TH-negative cell bodies and processes within the adrenergic C2 cell group area (Fig. 5). C3 cell group
Figure 6 shows the presence of weak, diffuse cytoplasmic NPY Y~ receptor-LI and strong cytoplasmic TH-LI nerve cell bodies in the adrenergic C3 cell group, located in the midline region immediately ventral to the fourth ventricle at the same level as groups CI and C2. A few TH-LI nerve cell body profiles were also scattered in the paramedian reticular nucleus. Eighty-one per cent of the TH-LI neurons showed NPY Y~ receptor-LI, which was almost exclusively located in TH-LI cell bodies, and specific NPY Y~ receptor-LI profiles were not seen in the TH-negative cell bodies and processes within the adrenergic C3 cell group area (Fig. 6, Table 1). DISCUSSION In the present study the localization of NPY receptors of the Y~ subtype in the AI, A2, C1, C2 and
523
C3 cell groups of the rat medulla oblongata has been indicated by means of a specific polyclonal antibody against the C-terminal portion of the rat NPY Y~ receptor. 69Thus, the control experiments suggest that the antibody can recognize the NPY receptors of the Y~ subtype, since preabsorption with the synthetic NPY Y~ receptor peptide (amino acids 355-382) can fully block the appearance of NPY Y~ receptor-LI in all the analysed areas. The TH immunostaining shows that the distribution and morphology of the TH-positive cell body profiles in the AI, A2, C1, C2 and C3 cell groups of the rat medulla oblongata observed in the present study are consistent with the description in previous studies. 38,39 In these cell groups, with the exception of the A2 cell group, the NPY Y~ receptor may work at least in part as an autoreceptor, in view of the existence of NPY-LI in these catecholaminergic cell groups. ~7However, these cell groups are also innervated by NPY-positive terminals, suggesting that they may also act as postsynaptic receptors. ~7 The major results of the present study are the demonstration of coexisting NPY Y1 receptor-LI and TH-LI in neurons of these areas. These findings, together with recent evidence for the coexistence of ~2A-adrenoceptor-LI and TH-LI in neurons of the A 1 cell group, 57 suggest that the NPY Y~ receptor may coexist with the ~2A-adrenoceptor in neurons of the A1 cell group. These TH-positive neurons, representing noradrenergic neurons containing NPY-LI, ~2'~7 project directly to cardiovascularly related nuclei such as the hypothalamic paraventricular nucleus, the nucleus tractus solitarii and the rostral ventrolateral medulla. 5'6'27'28'59'6°The neuroanatomical features of these noradrenergic neurons suggest an important role in the central cardiovascular control. 7,~° Stimulation of the A1 cell group results in vasodepressor and bradycardiac responses. 7'1° Moreover, the
Table 1. Co-localization of neuropeptide Y~ receptor-like immunoreactivity and tyrosine hydroxylase-like immunoreactivity in the A1, A2, C1, C2 and C3 cell groups of the rat medulla oblongata Number of c e l l s with TH-LI but without NPY Yj receptor-LI
Number of cells with both NPY Y~ receptor-LI and TH-LI
Total number of cells with TH-LI
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71
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128
130
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2
153
155
C2 cell group (Bregma -- 12.5 ram)
3
43
46
C3 cell group (Bregma -- l 2.5 ram)
3
13
16
One representative section from each brain at the same bregrna level was selected and the number of NPY Y Lreceptor-LI and TH-LI ceils was counted in a Nikon Microphot-FX. The cumulated counts from five rats are presented, n = 5.
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NPY Y~ receptor-LI in catecholaminergic neurons microinjections into the A 1 cell group of NPY and of clonidine, an ~2-adrenoceptor agonist and also an imidazoline receptor agonist, can mimic these cardiovascular effects. 45'48 These results indicate that NPY and c~2 and/or imidazoline receptors in this region mediate these cardiovascular effects. The present results give indications that these cardiovascular actions of NPY and clonidine may partly involve the A 1 noradrenergic neurons. Also, they give an important cellular substrate for possible interactions between NPY YI and ~2 receptors in the noradrenergic nerve cells of the A1 cell group. Recently, biochemical experiments have demonstrated that NPY can significantly attenuate the inhibitory effects of ~2-adrenoceptors on forskolin-stimulated cAMP levels in slices of the medulla oblongata including the A1 cell group. 47 The noradrenergic A2 cell group in the caudal part of the medial subnucleus of the nucleus tractus solitarii represents an important cardiovascular cell group. 3'9A1'12'38 Baroreceptor and chemoreceptor afferents terminate inter alia in the A2 cell group area and this group also interconnects with other cardiovascularly related nuclei? An outstanding fea-
ture of the nucleus tractus solitarii is the large quantity and diversity of neurotransmitters/ neuromodulators and their receptors localized in this area. 64 There exists evidence for interactions among these transmission lines in the nucleus tractus solitarii. 1'2°'22'23'3°'67'68Activation of NPY Yl receptors significantly reduces the affinity of ~2-adrenoceptor binding sites and counteracts the a2-adrenoceptor-mediated cardiovascular responses induced from the caudal and dorsal part of the nucleus tractus solitarii.67'6sThe present results show that the vast majority of the TH-LI nerve cells in the A2 cell group contain NPY Y~ receptor-LI. Therefore, this noradrenergic cell group can represent one important substrate for the above antagonistic NPY Y1/c~2 receptor-receptor interaction in the cardiovascular part of the nucleus tractus solitarii, since coexistence of NPY Y~ receptor-LI and TH-LI in noradrenergic neurons of the A2 cell group implies that these neurons possess both NPY Yl and ~2A receptors. Thus, a recent report shows that the noradrenergic neurons in the A2 area are labelled to a very high extent by a specific antibody to C~2A-adrenoceptors.57
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Fig. 6. Weak, diffuse cytoplasmic NPY Y1 receptor-LI and strong cytoplasmic TH-LI are shown in the adrenergic C3 cell group of the rat medulla oblongata by means of the double colour immunofluorescence technique. As seen in A and B, NPY Yl receptor-LI cell bodies (big arrows) show TH-LI (big arrowheads). Also, TH-LI dendrites (small arrowheads) exist in this area. Bregma level -12.5ham. Bar = 100/tm.
528
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The C1 cell g r o u p area located in the rostral ventrolateral medulla o b l o n g a t a is well recognized as a v a s o m o t o r centre. 26'31 It interconnects with a series of i m p o r t a n t cardiovascularly related nuclei a n d directly projects to the preganglionic sympathetic neurons in the intermediolateral cell c o l u m n of the spinal cord. 34'35~58"64The n e u r o n s in the CI cell g r o u p are adrenergic 35 a n d f o u n d to c o n t a i n ~2A-adrenoceptor-LI a n d N P Y - L I . ~7'57 In the present study, the adrenergic nerve cell bodies d e m o n s t r a t e d by T H - L I are also labelled by a specific a n t i b o d y against the N P Y YI receptor a n d thus a p p e a r to possess b o t h N P Y Y~ a n d ~2A-adrenergic receptors. The above cellular substrate with the existence of N P Y YI a n d ~2A-adrenergic receptors in n e u r o n s of the C1 cell g r o u p suggests that there m a y exist possible interactions between N P Y Y~ a n d ~2-adrenergic receptors in this cell group. However, the microinjection of N P Y at a dose o f 25 pmol into the CI cell g r o u p failed to m o d u l a t e cardiovascular effects induced by the ~z-adrenoceptor agonist a n d imidazoline receptor agonist clonidine. 48 Therefore, it m a y be considered t h a t u n d e r certain conditions including anaesthetized rats the imidazoline receptors but not ct2-adrenoce ptors mediate the hypotensive action of clonidine in the rostral ventrolateral medulla, t5"3255 It remains to be shown whether N P Y Y~ receptors a n d ~2-adrenoccptors interact with each other in the CI cell group. Also, the adrenergic n e u r o n s in the C2 a n d C3 cell groups have been observed to c o n t a i n ~2A-adrenoceptor-LI. In the present experiments, the N P Y Y~ receptor-LI is also localized in these adrenergic neurons of the C2 a n d C3 groups visualized by their presence o f TH-LI. 39 Thus, an interaction between N P Y Y~ a n d ~2-adrenergic receptors m a y also exist in the adrenergic C2 a n d C3 cell groups, k n o w n to contain N P Y - L I . ~v It should be noticed t h a t like the CI cell g r o u p the C2 a n d C3 cell groups directly
project to the intermediolateral cell column a n d to the nucleus tractus solitarii a n d c o n t r i b u t e to the regulation o f sympathetic outflow. 5~ F u r t h e r m o r e , the two cell groups are close to the fourth ventricle] 9 Therefore, the N P Y Y~ a n d ~2A receptors in the C2 a n d C3 cell groups m a y be involved in parts of the cardiovascular effects induced by intraventricular injections of adrenaline or NPY, 22 a l t h o u g h the m a j o r site of action m a y be the nucleus tractus solitarii, rich in the N P Y YI a n d ~2-adrenergic receptors. CONCLUSIONS By use of a polyclonal a n t i b o d y against the N P Y Y~ receptors a moderately intense, diffuse cytoplasmic N P Y Y~ receptor-LI has been d e m o n s t r a t e d in the noradrenergic A1 and A2 as well as in the adrenergic C1 and C2 cell groups, with the exception o f the C3 cell group containing only weak N P Y Y~-LI. These morphological results s u p p o r t the view t h a t N P Y YI receptors in the medulla o b l o n g a t a are involved in cardiovascular regulation, in view of the well-known cardiovascular role o f these catecholaminergic neurons, where they m a y coexist with the :t2A-adrenoceptor, previously d e m o n s t r a t e d in these neurons. The results provide a morphological substrate for the previously d e m o n s t r a t e d antagonistic N P Y Y~/c~2 r e c e p t o r - r e c e p t o r interactions in the nucleus tractus solitarii involved in cardiovascular control a n d point to a role o f the noradrenergic A2 n e u r o n s as one system involved in the vasodepressor action o f N P Y , n o r a d r e n a l i n e and adrenaline in the nucleus tractus solitarii. This work was supported by a grant from the Swedish Medical Research Council (04X-715), a grant from the Marianne and Marcus Wallenberg's Foundation and a grant from Glaxo, Verona, Italy. S.-N. Yang is supported by the Karolinska Institutet. Acknowledgements
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(1990) Neuropeptide Y and central cardiovascular regulation. Focus on its role as a cotransmitter in cardiovascular adrenergic neurons. Ann. N. Y. Acad. Sei. 611, 111 132. 21. Fuxe K., Bolme P., Agnati L. F., Jonsson G., Andersson K., K6hler C. and H6kfelt T. (1980) On the role of central adrenaline neurons in central cardiovascular regulation. In Central Adrenaline Neurons: Basic Aspects and their Role in Cardiovascular Disease (eds Fuxe K., Goldstein M., H6kfelt B. and H6kfelt T.), pp. 161-182. Pergamon Press, Oxford. 22. Fuxe K., Harfstrand A., Agnati L. F., Kalia M., Fredholm B., Svensson T., Gustafsson J. A., Lang R. and Ganten D. (1987) Central catecholamine neuropeptide Y interactions at the pre- and postsynaptic level in cardiovascular centers. J. cardiovase. Pharmac. 10, 1 13. 23. Fuxe K., Von Euler G., Van der Ploeg l., Fredholm B. B. and Agnati L. F. (1989) Pertussis toxin treatment counteracts the cardiovascular effects of neuropeptide Y and clonidine in the awake unrestrained rat. Neurosei. Lett. 101, 337-341. 24. Gehlert D. R. (1994) Subtypes of receptors for neuropeptide Y: implications for the targeting of therapeutics. Life Sci. 55, 551-562. 25. Gehlert D. R., Gackenheimer S. L. and Schober D. A. (1992) [Leu3~,Pro34] neuropeptide Y identifies a subtype of ~25I-labeled peptide YY binding sites in the rat brain. Neurochem. Int. 21, 45 67. 26. Giuliano R., Ruggiero D. A., Morrison S., Ernsberger P. and Reis D. J. (1989) Cholinergic regulation of arterial pressure by the C1 area of the rostral ventrolateral medulla. J. Neurosci. 9, 923 942. 27. Granata A. R., Kumada M. and Reis D. J. (1985) Sympathoinhibition by Al-noradrenergic neurons is mediated by neurons in the C1 area of the rostral medulla. J. auton. Nerv. Syst. I4, 387 395. 28. Granata A. R., Numao Y., Kumada M., Coulter J. D. and Reis D. J. (1986) AI noradrenergic neurons tonically inhibit sympathoexcitatory neurons of C1 area in rat brainstem. Brain Res. 377, 127 146. 29. Guyenet P. G., Stornetta R. L., Riley T., Norton F. R., Rosin D. L. and Lynch K. R. (1994) Alpha2A-adrenergic receptors are present in lower brainstem catecholaminergic and serotonergic neurons innervating spinal cord. Brain Res. 638, 285 294. 30. Hfirfstrand A., Fuxe K., Agnati L. and Fredholm B. (1989) Reciprocal interactions between ct2-adrenoceptor agonist and neuropeptide Y binding sites in the nucleus tractus solitarius of the rat. A biochemic and autoradiographic analysis. J. neural Transm. 75, 83 99. 31. Haselton J. R. and Guyenet P. G. (1989) Electrophysiological characterization of putative C1 adrenergic neurons in the rat. Neuroscienee 30, 199 214. 32. Heible P. J. and Ruffolo R. R. (1992) Imidazoline receptors: historical perspective. Fundam. clin. Pharmac. 6, 7-13. 33. Herzog H., Hort Y. J., Ball H. J., Hayes G., Shine J. and Selbie L. A. (1992) Cloned human neuropeptide Y receptor couples to two different second messenger systems. Proc. nam. Acad. Sei. U.S.A. 89, 5794-5798. 34. H6kfelt T., Foster G. A., Johansson O., Schultzberg M., Holets V., Ju G., Skagerberg G., Palkovits M., Skirboll L., Stolk J. M., U'Prichard D. C. and Goldstein M. (1988) Morphology of brain epinephrine systems. In Epinephrine in the Central Nervous System (eds Stolk J. M., U'Prichard D. C. and Fuxe K.), pp. 10 32. Oxford University Press, New York. 35. H6kfelt T., Fuxe K., Goldstein M. and Johansson O. (1974) Immunohistochemical evidence for the existence of adrenaline neurones in the rat brain. Brain Res. 66, 235-251. 36. Illes P. and Regenold J. T. (1990) Interaction between neuropeptide Y and noradrenaline on central catecholamine neurons. Nature 344, 62 63. 37. Jazin E. E., Zhang X., Soderstrom S., Williams R., H6kfelt T., Ebendal T. and Larhammar D. (1993) Expression of peptide YY and mRNA for the NPY/PYY receptor of the Y~ subtype in dorsal root ganglia during rat embryogenesis. Devl Brain Res. 76, I05 113. 38. Kalia M., Fuxe K. and Goldstein M. (1985) Rat medulla oblongata--ll. Dopaminergic, noradrenergic (A1 and A2) and adrenergic neurons, nerve fibers, and presumptive terminal processes. J. comp. Neurol. 233, 308-332. 39. Kalia M., Fuxe K. and Goldstein M. (1985) Rat medulla oblongata--IlI. Adrenergic (C1 and C2) neurons, nerve fibers and presumptive terminal processes. J. comp. Neurol. 233, 333 349. 40. Kassis S., Olasmaa M., Terenius L. and Fishman P. H. (1987) Neuropeptide Y inhibits cardiac adenylate cyclase through a pertussis toxin-sensitive G protein. J. biol. Chem. 262, 3429-3431. 41. Larhammar D., Blomqvist A. G., Yee F., Jazin E., Yoo H. and Wahlestedt C. (1992) Cloning and functional expression of a human neuropeptide Y/peptide YY receptor of the Y~ type. J. biol. Chem. 267, 10,935 10,938.
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42. Larsen P. J., Jessop D. S., Chowdrey H. S., Lightman S. L. and Mikkelsen J. D. (1994) Chronic administration of glucocorticoids directly upregulates prepro-neuropeptide Y and Y~-receptor mRNA levels in the arcuate nucleus of the rat. J. Neuroendoer. 6, 153 159. 43. Larsen P. J., Sheikh S. P., Jakobsen C. R., Schwartz T. W. and Mikkelsen J. D. (1993) Regional distribution of putative NPY Y~ receptors and neurons expressing Yj mRNA in forebrain areas of the rat central nervous system. Eur. J. Neurosci. 5, 1622 1637. 44. Leslie R. A., McDonald T. J. and Robertson H. A. (1988) Autoradiographic localization of peptide YY and neuropeptide Y binding sites in the medulla oblongata. Peptides 9, 1071 1076. 45. Macrae I. M. and Reid J. L. (1988) Cardiovascular significance of neuropeptide Y in the caudal ventrolateral medulla of the rat. Brain Res. 456, I 8. 46. Martel J. C., St Pierre S. and Quirion R. (1986) Neuropeptide Y receptors in rat brain: autoradiographic localization. Peptides 7, 55-60. 47. McAuley M. A., Macrae I. M., Farmer R. and Reid J. L. (1991) Effects of neuropeptide on forskolin, alpha 2- and beta-adrenoceptor-regulated cAMP levels in the rat brain slice. Peptides 12, 407 412. 48. McAuley M. A., Macrae I. M. and Reid J. L. (1989) The cardiovascular actions of clonidine and neuropeptide-Y in the ventrolateral medulla of the rat. Br. J. Pharmac. 97, 1067-1074. 49. Michel M. C. (1991) Receptors for neuropeptide Y: multiple subtypes and multiple second messengers. Trends pharmac. Sci. 12, 389-394. 50. Mikkelsen J. D. and Larsen P. J. (1992) A high concentration of NPY (Y0-receptor mRNA-expressing cells in the rat arcuate nucleus. Neurosci. Lett. 148, 195 198. 51. Minson J., Llewetlyn S. I., Neville A., Somogyi P. and Chalmers J. ([990) Quantitative analysis of spinally projecting adrenaline-synthesizing neurons of CI, C2 and C3 groups in rat medulla oblongata. J. auton. Nert,. Syst. 30, 209 220. 52. Nakajima M., Inui A., Teranishi A., Miura M., Hirosue Y., Okita M., Himori N., Baba S. and Kasuga M. (1994) Effects of pancreatic polypeptide family peptides on feeding and learning behavior in mice. J. Pharmac. exp. Ther. 268, 1010-1014. 53. Nicholas A. P., Pieribone V. and H6kfelt T. (1993) Distributions of mRNAs for alpha-2 adrenergic receptor subtypes in rat brain: an in situ hybridization study. J. comp. Neurol. 328, 575 594. 54. Paxinos G. and Watson C. (1986) The Rat Brain in Stereotaxic Coordinates, 2nd edn. Academic Press, Sydney. 55. Reis D. J., Regunathan S., Wang H., Feinstein D. U and Meeley M. P. (1992) lmidazoline receptors in the nervous system. Fundam. clin. Pharmae. 6, 23 29. 56. Rimland J., Xin W., Sweetnam P., Saijoh K., Nestler E. J. and Duman R. S. (1991) Sequence and expression of a neuropeptide Y receptor cDNA. Molee. Pharmac. 40, 869-875. 57. Rosin D. L., Zeng D., Stornetta R. L., Norton F. R., Riley T., Okusa M. D., Guyenet P. G. and Lynch K. R. (1993) Immunohistochemical localization of ~2A-adrenergic receptors in catecholaminergic and other brainstem neurons in the rat. Neuroscience 56, 139 155. 58. Ross C. A., Armstrong D. M., Ruggiero D. A., Pikel V. M., Loh T. H. and Reis D. J. (1981) Adrenaline neurones in rostral ventrolateral medulla innervate thoracic spinal cord: a combined immunocytochemical and retrograde transport demonstration. Neurosci. Lett. 25, 257 262. 59. Sakumoto T., Tohyama M., Satoh K., Kimoto Y., Kingasa T., Yanizawa O., Kurachi K. and Shimizu N. (1978) Afferent fiber connections from lower brainstem to hypothalamus studied by the horseradish peroxidase method with special reference to noradrenaline innervation. Expl Brain Res. 31, 81 90. 60. Sawchenko P. E. and Swanson L. W. (1982) The organisation of noradrenergic pathways from the brainstem to the paraventricular and supraoptic nuclei in the rat. Brain Res. Rev. 4, 275 325. 61. Stanley B. G. and Thomas W. J. (1993) Feeding responses to perifornical hypothalamic injection of neuropeptide Y in relation to circadian rhythms of eating behavior. Peptides 14, 475 481. 62. Tseng C. J., Mosqueda-Garcia R., Appalsamy M. and Robertson D. (1989) Cardiovascular effects of neuropeptide Y in rat brainstem nuclei. Circ. Res. 64, 55-61. 63. Unden A. and Bartfai T. (1984) Regulation of neuropeptide Y (NPY) binding by guanine nucleotides in the rat cerebral cortex. Fedn Eur. biochem. Sots Lett. 177, 125 128. 64. Van Giersbergen P. L. M., Palkovits M. and De Jong W. (1992) Involvement of neurotransmitters in the nucleus tractus solitarii in cardiovascular regulation. Physiol. Rev. 72, 789 824. 65. Von Euler G., Fuxe K., Van der Ploeg I., Fredholm B. B. and Agnati L. F. (1989) Pertussis toxin treatment counteracts intramembrane interactions between neuropeptide Y receptors and ~2-adrenoceptors. Eur. J. Pharmac. 172, 435 441. 66. Wan C. P. and Lau B. H. S. (1995) Neuropeptide Y receptor subtypes. Life Sci. 56, 1055-1064. 67. Yang S.-N., Fior D. R., Hedlund P. B., Agnati L. F. and Fuxe K. (1994) Antagonistic regulation of ~2-adrenoceptors by neuropeptide Y receptor subtypes in the nucleus tractus solitarii. Eur. J. Pharmac. 271, 201 212. 68. Yang S.-N., Fior D. R., Hedlund P. B., Narvaez J. A., Agnati L. F. and Fuxe K. (1994) Coinjections of NPY(I-36) or [Leu 3~,Pro34]NPY with adrenaline in the nucleus tractus solitarius of the rat counteract the vasodepressor responses to adrenaline. Neurosci. Lett. 171, 27 3t. 69. Zhang X., Bao L., Xu Z.-Q., Kopp J., Arvidsson U., Elde R. and H6kfelt T. (1994) Localization of neuropeptide Y Y~ receptors in the rat nervous system with special reference to somatic receptors on small dorsal root ganglion neurons. Proe. natn. Acad. Sci. U.S.A. 91, 11,738-11,742. (Accepted 29 January 1996)
Neuroscience Vol. 73, No. 2, pp. 519 530, 1996 Copyright © 1996 IBRO. Published by ElsevierScience Ltd Printed in Great Britain S0306-4522(96)00072-3 0306-4522/96 $15.00 + 0.00
LOCALIZATION OF NEUROPEPTIDE Y Y1 RECEPTOR-LIKE IMMUNOREACTIVITY IN CATECHOLAMINERGIC NEURONS OF THE RAT MEDULLA OBLONGATA S.-N. Y A N G , * t B. B U N N E M A N N , : ~ A. C I N T R A * and K. F U X E * *Division of Cellular and Molecular Neurochemistry, Department of Neuroscience, Karolinska Institutet, Doktorsringen 12, S-171 77 Stockholm, Sweden ++Department of Pharmacology, Glaxo Research Laboratory, Verona, Italy Abstract--Neuropeptide Y receptors in the medulla oblongata participate in central cardiovascular control. The neuropeptide Y~ receptor subtype gene and amino acid sequence have been identified by molecular cloning studies. In this study, a C-terminal peptide representing amino acids 355-382 of the neuropeptide Yt receptor was synthesized and cross-linked to thyroglobulin to produce an antibody against a partial sequence of the neuropeptide Y~ receptor, used to localize neuropeptide Y~ receptor-like immunoreactivity in the catecholaminergic neurons of the medulla oblongata. The double colour immunofluorescence technique with a polyclonal antibody against the neuropeptide Yt receptor and a monoclonal antibody against tyrosine hydroxylase revealed that in the rat medulla oblongata, a weak (the C3 cell group) to moderately intense (the A 1, A2, C 1 and C2 cell groups), diffuse cytoplasmic neuropeptide Y~ receptor-like immunoreactivity was distributed primarily in the noradrenergic and adrenergic cell bodies and occasionally seen in the noradrenergic and adrenergic cell processes. Almost all tyrosine hydroxylase-like immunoreactive cell bodies in the AI, A2, CI, C2 and C3 cell groups showed neuropeptide Yt receptor-like immunoreactivity. The neuropeptide YI receptor-like immunoreactivity in the A2 cell group was somewhat stronger. The present findings show localization of specific neuropeptide Yt receptor-like immunoreactivity in the vast majority of the noradrenergic and adrenergic cell bodies of the A1, A2, CI, C2 and C3 cell groups, which are putative cardiovascular regions. The results support the view that neuropeptide Y~ receptors in the medulla oblongata are involved in central cardiovascular control and may coexist with another important receptor, the %A-adrenoceptor, also involved in central cardiovascular regulation, since the cqA-adrenoceptor-like immunoreactivity has been shown to exist in almost all noradrenergic and adrenergic cell bodies in the brainstem. In conclusion, centrally administered neuropeptide Y may act in part via neuropeptide Y~ receptors located on the soma and dendrites of noradrenergic and adrenergic neurons, where it may interact with c~2-adrenoceptors at least in the noradrenergic A2 neurons. This noradrenaline system may be involved in at least part of the vasodepressor actions of neuropeptide Y, noradrenaline and adrenaline in the nucleus tractus solitarii in view of the present findings. Copyright .© 1996 IBRO. Published by Elsevier Science Ltd. Key words: medulla oblongata, adrenergic, noradrenergic, peptide receptor, immunocytochemistry, tyrosine hydroxylase.
effects. 24'49'66 However, only the N P Y YI receptor gene has been cloned) 6"3s'41'56 It has been shown that the N P Y YI receptor has seven transmembrane domains, three extracellular and three intracellular loops like other members of the G-protein-coupled superfamily of receptors. 16'18'19'33'4°'49'63 In situ hybridization studies have demonstrated that N P Y Yj receptor m R N A s are strongly expressed in peripheral and central neurons. 37'42'43'5°'69The adrenergic and noradrenergic nuclei in the medulla oblongata responsible for regulating cardiovascular activity 3'8'9"H'21 contain a high density of N P Y Y~ receptor binding sites. 2'25'44,46 However, autoradiographical evidence suffers from the lack of cellular resolution and thus does not establish the existence of binding sites on neurons and/or on glia,
Neuropeptide Y (NPY) is a putative regulatory peptide in the brain and participates in the regulation of several physiological functions including the autonomic activity, especially cardiovascular control, food intake, circadian rhythm, m e m o r y and neuroendocrine regulation. 4A3'14'52'61'62 These physiological actions of N P Y depend on the activation of N P Y receptors. Five N P Y receptor subtypes, YI, Y2, Y3, " F e e d i n g " and " P Y Y preferring", have been identified according to their pharmacological properties and physiological tTo whom correspondence should be addressed. -LI, -like immunoreactivity or immunoreactive; NPY, neuropeptide Y; PBS, phosphatebuffered saline; TH, tyrosine hydroxylase.
Abbreviations:
519
520
S.-N. Yang et al.
and does not identify the neurochemical features of cells with N P Y YI receptor binding sites. N P Y Y~ receptors do not always achieve their physiological effects independently. They often do so interacting with other receptors such as ~2-adrenoceptors and angiotensin II receptors involving G-protein-dependent intramembrane recepto~receptor interactions. 1"2°'22'23"3°'36"65"67'68 The findings from this laboratory indicate that activation of N P Y YI receptors in the nucleus tractus solitarii can significantly decrease the affinity of high-affinity c~2-adrenoceptor binding sites and substantially counteract the ~2-adrenoceptor-mediated vasodepressor and bradycardic responses in the nucleus tractus solitarii. 6768 On the basis of these findings it can be postulated that the N P Y Y~ receptor may be localized together with e2-adrenoceptors in neurons of the nucleus tractus solitarii. More recent evidence indicates that e2Aadrenergic receptor-like immunoreactivity (-LI) is present in almost all noradrenergic and adrenergic cells of the brainstem. 29"53-57 In the present study an antibody against a C-terminal portion of the N P Y Y~ receptor (see Ref. 69) was employed to study the possible coexistence of NPY Y~ receptor-LI and tyrosine hydroxylase-LI (TH-LI) in the A1, A2, CI, C2 and C3 cell groups in the medulla oblongata in order to identify the neurochemical features of neurons with N P Y Yj receptor-LI in the medulla oblongata. Such a study will also indirectly give indications on the co-localization of N P Y Y~ receptors and ~2-adrenoceptors. Therefore there exists a potential to obtain an important morphological substrate for NPY Y~/e2 receptor~receptor interactions in the nucleus tractus solitarii known to be involved in cardiovascular c o n t r o l . 67,68
EXPERIMENTAL PROCEDURES
Animals
Male specific pathogen-free Sprague Dawley rats (180 200g body weight; B & K Universal, Stockholm, Sweden) were used. They were given food pellets and water ad libitum and kept under standardized temperature, humidity and lighting conditions (lights on at 8.00 a.m. and off at 8.00 p.m.).
Leitz cryostat at the analysed bregma level according to the atlas of Paxinos and Watson? 4 The sections were collected in the test tube and processed by the two colour immunofluorescence technique with the free-floating method. The sections were incubated for 48 h with a mixture of the rabbit antibody against the NPY Y~ receptor (1:5000 in 0.1 M PBS/0.5% Triton X-100) and the monoclonal mouse antibody against rat TH (1:800 in 0.1 M PBS/0.5% Triton X-100; INCSTAR Co. Minnesota, U.S.A.). After washing 3 x l0 min in 0.1 M PBS, the sections were incubated for 4 h at room temperature with a mixture of fluoresceine isothiocyanate-conjugated donkey anti-mouse immunoglobulins (1:40 in 0.1 M PBS/0.5% Triton X-100, Jackson, West Grove, PA) and CY3VM-conjugated AffiniPure goat antirabbit IgG (1 : 100 in 0.1 M PBS/0.5% Triton X-100, Jackson, West Grove, PA). The sections were then rinsed three times for 10min in 0.1 M PBS and finally mounted in an antifading medium and examined in a Nikon Microphot-FX epifluorescence microscope equipped with the appropriate excitation and barrier filter combinations. The specificity of the antibody was tested by absorption with the synthetic C-terminal peptide (amino acids 355 382) in a concentration of 20/*g/ml diluted antibody. One representative section from each brain at the same bregma level was selected and examined in a Nikon Microphot-FX microscope with a x 10 or x20 objective depending upon the area. The number of NPY Y~ receptorLI and TH-Li cells was counted within the AI, A2, CI, C2 and C3 celI groups, respectively. The cumulated counts from five rats were reported.
RESULTS
A moderately intense, diffuse cytoplasmic N P Y Yl receptor-LI was observed in the noradrenergic AI and A2 cell groups as well as in the adrenergic C1 and C2 cell groups, being somewhat stronger in the A2 cell group. A weak, diffuse cytoplasmic NPY Yl receptor-LI was present in the adrenergic C3 cell group. The appearance of the immunoreactivity was fully blocked by preabsorption of the antibody with the synthetic C-terminal peptide representing a partial sequence of the N P Y YI receptor. As seen in Fig. 1A, a diffuse cytoplasmic NPY Y~ receptor-LI existed in the noradrenergic A2 cell group area, but no NPY Yl receptor-LI was observed in the same area of an adjacent section preabsorbed with the synthetic C-terminal peptide (20/~g/ml diluted antibody; Fig. 1B). The same results were observed in the A I , C1, C2 and C3 cell groups.
Preparation (~["antibodies
A C-terminal peptide representing amino acids 355 382 of the NPY Y~ receptor was synthesized, cross-linked to thyroglobulin and injected into two rabbits. Sera were collected after two boosters and then purified. 69 Immunoeytochemist O,
Five rats were anaesthetized with sodium pentobarbital (60mg/kg, i.p.). The transcardiac perfusion was then performed by using a fixative consisting of 2% paraformaldehyde and 14% v/v saturated picric acid in 0.1 M sodium phosphate buffer (pH 7.3). The brain was rapidly dissected and postfixed by immersion in the fixative solution for 90min and subsequently washed twice in 0. I M phosphate-buffered saline (PBS) for 10min. The brains were then placed in 0.1 PBS containing 20% sucrose for 24h. Coronal sections (50/~m thick) were cut with a
A 1 cell group
A prominent cytoplasmic T H - L I and moderately intense, diffuse cytoplasmic N P Y Y~ receptor-LI was observed in the noradrenergic A1 cell group area of the caudal medulla oblongata, located in the ventrolateral part of the medullary reticular formation. As seen in Fig. 2, the N P Y Y~ receptor-Ll was exclusively present in T H - L I cell bodies and in dendritic processes. Ninety-six per cent of the T H - L I neurons were positive for N P Y Y~ receptor-LI and specific N P Y Y~ receptor-LI profiles were not present in the TH-negative cell bodies and processes within the noradrenergic A1 cell group area (Fig. 2, Table I).
NPY YL receptor-LI in catecholaminergic neurons A 2 cell group
As shown in Fig. 3, a cytoplasmic TH-LI of strong intensity and diffuse cytoplasmic NPY Y1 receptor-LI of moderate intensity appeared in the noradrenergic A2 cell group area of the medial subnucleus of the nucleus tractus solitarii. The TH-LI was also seen in the nerve terminals. A few TH-LI cell body profiles were scattered in the dorsal motor nucleus of the vagus, the intermediate subnucleus of the nucleus tractus solitarii and the commissural nucleus. The NPY Y~ receptor-Ll was almost exclusively present in TH-LI cell bodies. A few TH-LI dendrites were also NPY Y~ receptor positive. The vast majority of TH-LI cells (98%) were double-labelled (Table 1) and specific NPY Y~ receptor-LI profiles
A
Gr
521
were not seen in the TH-negative cell bodies and processes within the noradrenergic A2 cell group area (Fig. 3). C I cell group
Moderately intense, diffuse cytoplasmic NPY Yt receptor-LI and strong cytoplasmic TH-LI cell body profiles appeared in the adrenergic C1 cell group, mainly found in the rostral ventrolateral part of the medullary reticular formation. The NPY Yj receptorLI was exclusively found in TH-LI cell bodies and in dendritic processes. Ninety-nine per cent of the THLI neurons were double-labelled with the NPY Y1 receptor antibody (Fig. 4, Table 1). Moreover, specific NPY Yl receptor-LI profiles were not
NpY Y1-L| AP
¢¢
¢ / t
E3
Gr
¢
Preabsorption AP
NTS
Fig. I. A substantial number of cell body profiles with diffuse cytoplasmic NPY Y1 receptor-LI (arrows) of moderate intensity exists in the noradrenergic A2 cell group area (A), but no NPY Y~ receptor-LI (B) is seen in the same area of the adjacent section, where the NPY Yt receptor antibody has been preabsorbed with the synthetic peptide, representing a partial sequence of the NPY Yt receptor. The indirect immunofluorescencetechnique has been used. AP, area postrema; Gr, gracile nucleus; NTS, the nucleus tractus solitarii; asterisk, central canal. Bregma level -14.0 mm. Bar = 100 #m.
522
i~~
S.-N. Y a n g et al.
~
<~
o=~lr
~.~. ~=
.~
.=-
~2
NPY Y~ receptor-Ll in catecholaminergic neurons observed in the TH-negative cell bodies and processes within the adrenergic C1 cell group area (Fig. 4). C2 cell group
In this adrenergic cell group, cell body profiles with diffuse cytoplasmic NPY Y~ receptor-LI of moderate intensity and with cytoplasmic TH-LI of strong intensity were found on the dorsal border of the rostral nucleus tractus solitarii. A dense network of TH-LI terminals appeared in the medial pole of the nucleus tractus solitarii. The NPY Y~ receptor-LI was almost exclusively observed in TH-LI cell bodies. The percentage co-localization of TH-LI and NPY Y1 receptor-LI was 93% in relation to TH-LI neurons (Fig. 5, Table 1). Specific NPY YI receptor-LI profiles did not appear in the TH-negative cell bodies and processes within the adrenergic C2 cell group area (Fig. 5). C3 cell group
Figure 6 shows the presence of weak, diffuse cytoplasmic NPY Y~ receptor-LI and strong cytoplasmic TH-LI nerve cell bodies in the adrenergic C3 cell group, located in the midline region immediately ventral to the fourth ventricle at the same level as groups CI and C2. A few TH-LI nerve cell body profiles were also scattered in the paramedian reticular nucleus. Eighty-one per cent of the TH-LI neurons showed NPY Y~ receptor-LI, which was almost exclusively located in TH-LI cell bodies, and specific NPY Y~ receptor-LI profiles were not seen in the TH-negative cell bodies and processes within the adrenergic C3 cell group area (Fig. 6, Table 1). DISCUSSION In the present study the localization of NPY receptors of the Y~ subtype in the AI, A2, C1, C2 and
523
C3 cell groups of the rat medulla oblongata has been indicated by means of a specific polyclonal antibody against the C-terminal portion of the rat NPY Y~ receptor. 69Thus, the control experiments suggest that the antibody can recognize the NPY receptors of the Y~ subtype, since preabsorption with the synthetic NPY Y~ receptor peptide (amino acids 355-382) can fully block the appearance of NPY Y~ receptor-LI in all the analysed areas. The TH immunostaining shows that the distribution and morphology of the TH-positive cell body profiles in the AI, A2, C1, C2 and C3 cell groups of the rat medulla oblongata observed in the present study are consistent with the description in previous studies. 38,39 In these cell groups, with the exception of the A2 cell group, the NPY Y~ receptor may work at least in part as an autoreceptor, in view of the existence of NPY-LI in these catecholaminergic cell groups. ~7However, these cell groups are also innervated by NPY-positive terminals, suggesting that they may also act as postsynaptic receptors. ~7 The major results of the present study are the demonstration of coexisting NPY Y1 receptor-LI and TH-LI in neurons of these areas. These findings, together with recent evidence for the coexistence of ~2A-adrenoceptor-LI and TH-LI in neurons of the A 1 cell group, 57 suggest that the NPY Y~ receptor may coexist with the ~2A-adrenoceptor in neurons of the A1 cell group. These TH-positive neurons, representing noradrenergic neurons containing NPY-LI, ~2'~7 project directly to cardiovascularly related nuclei such as the hypothalamic paraventricular nucleus, the nucleus tractus solitarii and the rostral ventrolateral medulla. 5'6'27'28'59'6°The neuroanatomical features of these noradrenergic neurons suggest an important role in the central cardiovascular control. 7,~° Stimulation of the A1 cell group results in vasodepressor and bradycardiac responses. 7'1° Moreover, the
Table 1. Co-localization of neuropeptide Y~ receptor-like immunoreactivity and tyrosine hydroxylase-like immunoreactivity in the A1, A2, C1, C2 and C3 cell groups of the rat medulla oblongata Number of c e l l s with TH-LI but without NPY Yj receptor-LI
Number of cells with both NPY Y~ receptor-LI and TH-LI
Total number of cells with TH-LI
A I cell group (Bregma - 14.3 ram)
3
68
71
A2 cell group (Bregma - 14.0 mm)
2
128
130
C I cell group (Bregma -- 12.5 mm)
2
153
155
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NPY Y~ receptor-LI in catecholaminergic neurons microinjections into the A 1 cell group of NPY and of clonidine, an ~2-adrenoceptor agonist and also an imidazoline receptor agonist, can mimic these cardiovascular effects. 45'48 These results indicate that NPY and c~2 and/or imidazoline receptors in this region mediate these cardiovascular effects. The present results give indications that these cardiovascular actions of NPY and clonidine may partly involve the A 1 noradrenergic neurons. Also, they give an important cellular substrate for possible interactions between NPY YI and ~2 receptors in the noradrenergic nerve cells of the A1 cell group. Recently, biochemical experiments have demonstrated that NPY can significantly attenuate the inhibitory effects of ~2-adrenoceptors on forskolin-stimulated cAMP levels in slices of the medulla oblongata including the A1 cell group. 47 The noradrenergic A2 cell group in the caudal part of the medial subnucleus of the nucleus tractus solitarii represents an important cardiovascular cell group. 3'9A1'12'38 Baroreceptor and chemoreceptor afferents terminate inter alia in the A2 cell group area and this group also interconnects with other cardiovascularly related nuclei? An outstanding fea-
ture of the nucleus tractus solitarii is the large quantity and diversity of neurotransmitters/ neuromodulators and their receptors localized in this area. 64 There exists evidence for interactions among these transmission lines in the nucleus tractus solitarii. 1'2°'22'23'3°'67'68Activation of NPY Yl receptors significantly reduces the affinity of ~2-adrenoceptor binding sites and counteracts the a2-adrenoceptor-mediated cardiovascular responses induced from the caudal and dorsal part of the nucleus tractus solitarii.67'6sThe present results show that the vast majority of the TH-LI nerve cells in the A2 cell group contain NPY Y~ receptor-LI. Therefore, this noradrenergic cell group can represent one important substrate for the above antagonistic NPY Y1/c~2 receptor-receptor interaction in the cardiovascular part of the nucleus tractus solitarii, since coexistence of NPY Y~ receptor-LI and TH-LI in noradrenergic neurons of the A2 cell group implies that these neurons possess both NPY Yl and ~2A receptors. Thus, a recent report shows that the noradrenergic neurons in the A2 area are labelled to a very high extent by a specific antibody to C~2A-adrenoceptors.57
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Fig. 6. Weak, diffuse cytoplasmic NPY Y1 receptor-LI and strong cytoplasmic TH-LI are shown in the adrenergic C3 cell group of the rat medulla oblongata by means of the double colour immunofluorescence technique. As seen in A and B, NPY Yl receptor-LI cell bodies (big arrows) show TH-LI (big arrowheads). Also, TH-LI dendrites (small arrowheads) exist in this area. Bregma level -12.5ham. Bar = 100/tm.
528
S.-N. Yang et al.
The C1 cell g r o u p area located in the rostral ventrolateral medulla o b l o n g a t a is well recognized as a v a s o m o t o r centre. 26'31 It interconnects with a series of i m p o r t a n t cardiovascularly related nuclei a n d directly projects to the preganglionic sympathetic neurons in the intermediolateral cell c o l u m n of the spinal cord. 34'35~58"64The n e u r o n s in the CI cell g r o u p are adrenergic 35 a n d f o u n d to c o n t a i n ~2A-adrenoceptor-LI a n d N P Y - L I . ~7'57 In the present study, the adrenergic nerve cell bodies d e m o n s t r a t e d by T H - L I are also labelled by a specific a n t i b o d y against the N P Y YI receptor a n d thus a p p e a r to possess b o t h N P Y Y~ a n d ~2A-adrenergic receptors. The above cellular substrate with the existence of N P Y YI a n d ~2A-adrenergic receptors in n e u r o n s of the C1 cell g r o u p suggests that there m a y exist possible interactions between N P Y Y~ a n d ~2-adrenergic receptors in this cell group. However, the microinjection of N P Y at a dose o f 25 pmol into the CI cell g r o u p failed to m o d u l a t e cardiovascular effects induced by the ~z-adrenoceptor agonist a n d imidazoline receptor agonist clonidine. 48 Therefore, it m a y be considered t h a t u n d e r certain conditions including anaesthetized rats the imidazoline receptors but not ct2-adrenoce ptors mediate the hypotensive action of clonidine in the rostral ventrolateral medulla, t5"3255 It remains to be shown whether N P Y Y~ receptors a n d ~2-adrenoccptors interact with each other in the CI cell group. Also, the adrenergic n e u r o n s in the C2 a n d C3 cell groups have been observed to c o n t a i n ~2A-adrenoceptor-LI. In the present experiments, the N P Y Y~ receptor-LI is also localized in these adrenergic neurons of the C2 a n d C3 groups visualized by their presence o f TH-LI. 39 Thus, an interaction between N P Y Y~ a n d ~2-adrenergic receptors m a y also exist in the adrenergic C2 a n d C3 cell groups, k n o w n to contain N P Y - L I . ~v It should be noticed t h a t like the CI cell g r o u p the C2 a n d C3 cell groups directly
project to the intermediolateral cell column a n d to the nucleus tractus solitarii a n d c o n t r i b u t e to the regulation o f sympathetic outflow. 5~ F u r t h e r m o r e , the two cell groups are close to the fourth ventricle] 9 Therefore, the N P Y Y~ a n d ~2A receptors in the C2 a n d C3 cell groups m a y be involved in parts of the cardiovascular effects induced by intraventricular injections of adrenaline or NPY, 22 a l t h o u g h the m a j o r site of action m a y be the nucleus tractus solitarii, rich in the N P Y YI a n d ~2-adrenergic receptors. CONCLUSIONS By use of a polyclonal a n t i b o d y against the N P Y Y~ receptors a moderately intense, diffuse cytoplasmic N P Y Y~ receptor-LI has been d e m o n s t r a t e d in the noradrenergic A1 and A2 as well as in the adrenergic C1 and C2 cell groups, with the exception o f the C3 cell group containing only weak N P Y Y~-LI. These morphological results s u p p o r t the view t h a t N P Y YI receptors in the medulla o b l o n g a t a are involved in cardiovascular regulation, in view of the well-known cardiovascular role o f these catecholaminergic neurons, where they m a y coexist with the :t2A-adrenoceptor, previously d e m o n s t r a t e d in these neurons. The results provide a morphological substrate for the previously d e m o n s t r a t e d antagonistic N P Y Y~/c~2 r e c e p t o r - r e c e p t o r interactions in the nucleus tractus solitarii involved in cardiovascular control a n d point to a role o f the noradrenergic A2 n e u r o n s as one system involved in the vasodepressor action o f N P Y , n o r a d r e n a l i n e and adrenaline in the nucleus tractus solitarii. This work was supported by a grant from the Swedish Medical Research Council (04X-715), a grant from the Marianne and Marcus Wallenberg's Foundation and a grant from Glaxo, Verona, Italy. S.-N. Yang is supported by the Karolinska Institutet. Acknowledgements
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