Naunyn-Schmiedeberg's Arch Pharmacol (1984) 326: 222-226 Naunyn-Schmiedeberg's

Archives of Pharmacology �9 Springer-Verlag 1984

Intracerebral injection of different antibodies against endogenou s opioids suggests a-neoendorphin participation in control of feeding behaviour Riidiger Schulz 1, Annemarie Wilhelm 1, and Gerhard Dirlich 2 1 Max-Planck-Institut fiir Psychiatric, Abteilung ftir Neuropharmakologie, Am Klopferspitz 18 a, D-8033 Planegg-Martinsried 2 Max-Planck-Institut fiir Psychiatric, Abteilung ffir Biostatistiks, Kraepelinstrasse 2, D-8000 Miinchen 40, Federal Republic of Germany

Summary. The mechanism of feeding behaviour of rats was examined. We used antibodies to different opioid peptides in order to reduce the tonic activity of various endogenous opioid peptide systems that may underly appetite. Unilateral microinjection of anti-~-neoendorphin antibodies into various areas of the ventromedial hypothalamus (VMH) inhibited food and water intake up to 45% in deprived animals. Injections outside this area failed to affect feeding. Administration of anti-fl-endorphin antibodies into the VMH moderately attenuated appetite. A considerable decrease of food and water intake was observed only upon injection of this antibody into the nucleus periventricularis hypothalami, a region generally believed to be involved with feeding. A marginal reduction of appetite was observed with anti-dynorphin antibodies injected into the VMH. These data may suggest that c~-neoendorphin is involved in the control of food and water intake in the VMH.

Key words: Antiserum - Opioid peptides - Feeding be- haviour - c~-Neoendorphin

Introduction

Endogenous opioid peptides are supposed to be involved in feeding behaviour, since narcotic antagonists have been repeatedly shown to decrease food and water intake in experimental animals. This notion is consistent with findings that certain opioids give rise to eating when injected into specific areas of the brain (for reviews see Morley 1980; Sanger 1981). However, no information is available regard- ing the type of endogenous ligand of the opioid receptors associated with the physiological control of feeding.

A series of different opioid peptides discovered during recent years are present in the ventromedial hypothalamus (VMH) (Dupont et al. 1980; Goldstein and Ghazarossian 1980; Minamino et al. 1981 ; Maysinger et al. 1982), an area of the brain involved in feeding behaviour (Morgane 1961). One is ]~-endorphin which caused food intake when injected into the VMH (Grandison and Guidotti 1977) or into the

�9 lateral cerebral ventricle (McKay et al. 198l). It is also seen that decreased ]~-endorphin levels occur in the hypothalamus during fasting (Gambert et al. 1980). Even more interest has been directed on the ~c-opioid receptor ligands. Although

Send offprint requests to R. Schulz at the above address

administration of the prototype lc-agonist ethylketazocine into the VMH failed to affect feeding (Tepperman and Hirst 1982), several other investigations using either tc-agonists or antagonists strongly suggested the involvement of tc-ligands in the control of food and water intake (Morley and Levine 1981; Morley et al. 1982; Locke et al. 1982; Leander and Hynes 1983). Dynorphin and a-neoendorphin are found in the hypothalamus and both have been demonstrated to combine with ~c-opioid receptors or 3- and ~c-receptors (Wfister et al. 1981 a, b; Chavkin et al. 1982; Oka et al. 1982; Schulz et al. 1982).

The present studies were undertaken to gain more insight into the issue of which type of endogenous opioid may be involved in appetite. It is known that naloxone causes a decrease in food and water intake, presumably by blockade of tonic inhibition by opioid mechanisms (Morley 1980). We hypothesized that a similar effect to that obtained by blocking opioid receptors with naloxone would be achieved by precipitating a specific endogenous opioid in the vicinity of its receptor, using antibodies against different opioid peptides. An antiserum being effective to reduce appetite would be indicative for the specific endogenous opioid re- gulating the feeding mechanism. The antisera employed were directed against fi-endorphin, dynorphin and c~-neo- endorphin.

Methods

Male Sprague-Dawley rats (260-280 g) were employed. Each animal was implanted using stereotaxic procedures with a permanent stainless steel guide cannula directed into the right VMH. For technical details see Laschka et al. (1976). The next day the animals were housed singly, and were put on a restricted feeding regimen. Rats only received ad libitum food (Altromin standard diet) and tap water between 10.00 and 16.30 h. Lights were maintained on a 14 h on, 10 h off light/dark cycle, with lights on at 07.00 h and off at 21.00 h. The quantity of food and water consumed daily was recorded for each rat. After 14 days the intake had stabilized and experimentation started. A 5 I11 Hamilton syringe (the tip was square with an outer diameter 0.44 ram) was employed to inject the test solution (1 lal) through the guide cannula into conscious rats. The injection was carried out between 08.45 and 09.15 h. The unilateral injections were conducted over a 3 min period and the needle was left in place for a further 3 rain. Thereafter, the rats were re- turned to their cages and food and water intake was recorded for the 10.00 to 16.30 h period. Each rat was used only once.

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At the end of the experiment the locus of injection was verified by histological examination according to the atlas of K6nig and Klippel (1963).

The following antisera were employed: anti-/Lendorphin (titre 1:50000; that is, 50% binding of 15 fmol 12sI tracer), anti-c~-neoendorphin (1:25000) and anti-dynorphin A1-13 (1:20000, "Agathe"). The antisera were obtained and partially characterized as described elsewhere (H611t et al. 1979, 1980; Maysinger et al. 1982). Briefly, the anti-/% endorphin antibodies completely crossreacted with/~-lipo- tropin. However, the antiserum failed to interact with up to 10 nmole per tube of enkephalins, c~-neoendorphin or dynorphin A1-13. The major site of antidynorphin anti- serum recognition was the C-terminal part in dynorphin A1 ~3 which explains its inability to bind to/~-endorphin, the enkephalins or ~-neoendorphin. These antibodies com- pletely cross-reacted with dynorphin A. Anti-c~-neo- endorphin antiserum recognizes equally well /%neoendor- phin, but fails to bind to/%endorphin, the enkephalins and dynorphin A. As the titres of the anti-dynorphin antiserum ("Agathe") and the anti-c~-neoendorphin antiserum were re- latively low, the respective immunoglobulins were isolated by means of protein A-Sepharose (Ey et al. 1978) and con- centrated to a final titre of 1 : 80000 for the anti-dynorphin antiserum ("Agathe"), and 1 : 40000 for anti-~-neoendorphin antiserum. The concentrated anti-~-neoendorphin anti- bodies displayed identical cross-reactivity characteristics as described for the native antiserum. 1 gl anti-/~-endorphin antiserum was able to bind 6 pmol ~25I-/3-endorphin. The corresponding data were 8 pmol for the concentrated anti- dynorphin A antibodies, and 5 pmol for the concentrated c~-neoendorphin antibodies. Cyanogen bromide-activated Sepharose 4B coupled to /%endorphin, dynorphin A1_13 and c~-neoendorphin, respectively, was used to remove selectively the antibodies from the anti-/%endorphin anti- serum or the solutions containing the concentrated anti- dynorphin A or anti-e-neoendorphin antibodies. These solutions completely lost their binding capacity and served as controls. Few controls were also run with serum of rabbits before they were immunized with dynorphin A~-~3 and c~-neoendorphin, respectively.

The effect of the antibody microinjections on the food and water intake was analyzed for each rat and expressed as percent change of the mean values of the food and water intake of the two preceding days. The general behaviour of microinjected rats was noted and compared to that of untreated animals trained under identical conditions. These observations revealed no apparent difference over a period of about 4 h. Thereafter the injected rats appeared some- what less active, but this occurred regardless of whether they received the antibodies or the control material. Pilot experiments with I 0-fold lower amounts of c~-neoendorphin antibodies microinjected did not reveal significant changes in feeding behaviour.

For tests of significance of the treatment the Mann- Whitney U-test was employed.

Results

Rats adapted for two weeks to the feeding schedule ex- hibited an average food and water intake for the last two days of 21 _+ 3.1 g (n = 84) diet and 26 _+ 4.8 ml (n = 103) water. Only those rats showing a variability for these

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Fig. 1. The effect of microinjections of control solutions (control right panel) and anti-e-neoendorphin antibodies (ANE AB, left panel) into defined areas of the hypothalamus on food (�9 and water (O) intake. The data of one rat correspond to a single symbol for food and water intake. The percentage increase (+) or decrease ( - ) for the respective parameter is given on the abscissa. The hypothalamic areas microinjected are indicated on the ordinate, abbreviated according to K6nig and Klippel (1963): hpv, nucleus periventricularis; hvmm, nucleus ventromedialis, pars medialis; hvml, nucleus ventromedialis, pars lateralis; hdv, nucleus dorso- medialis, pars ventralis; ar, nucleus arcuatus; fp, nucleus paraventricularis (filiformis), pars parvocellularis; mml, nucleus mamillaris medialis, pars lateralis; fm, nucleus paraventricularis (filiformis), pars magnocellularis; hdd, nucleus dorsomedialis, pars dorsalis; ha, nucleus anterior; pv, nucleus premamillaris ventralis; re, nucleus reuniens; FMP, fasciculus medialis prosencephali; FMT, fasciculus mamillothalamicus; ZI, zona incerta

parameters of less than 10% were employed for experiment- ation.

The effect of control injections into different areas of the hypothalamus on food and water consumption are pre- sented on the right part of Fig. 1 (abbreviations given on the ordinate). The data of all control injections obtained for the different antisera have been combined, since no systematic differences were observed. The data of the control groups for all areas tested are randomly distributed around 0%. The left panel demonstrates the effect of anti-c~- neoendorphin antibodies. Injections into areas confined to the VMH (hpv, hvmm, hvml, hdv, ar, fp, mml, fro) con- sistently caused a reduction of food and water consumption of about 45%. A comparison of the individual areas with their respective controls reveals that the effect is highly sig- nificant for both parameters (P < 0.01) and for most of the areas examined. A considerable decrease of food and water intake was also observed in the areas ar, mml and fin, but the sample size was too small for statistical testing.

In order to examine a possible relationship between the reduction of food and of water consumption produced by anti-~-neoendorphin antibodies, the correlation coefficient

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Fig. 2. The relationship between food and water consumption in rats microinjected with anti-~-neoendorphin antibodies into the VMH. The abscissa represents percent decrease in water intake, the ordinate indicates percent decrease in food intake.�9 Each point consists of data obtained from one animal. The regression line is defined by y = -31.2 + 0.43 �9 x

for the data from eight areas of the VMH (hpv, hvmm, hvml, hdv, ar, fp, mml, fro; Fig. 2) was computed. It was shown to be 0.466. This value suggests some relationship between the reduction of food and water intake. Their changes ap- pear to be positively correlated and they are approximately proportional. The computed regression line is depicted in Fig. 2 and illustrates the linear relationship between food and water intake. However, these data do not provide a sufficient basis for a detailed examination of the type of relationship. Those animals receiving these antibodies into areas outside (re, FMP, FMT, ZI) or marginal (hdd, ha, pv) to the VMH responded not differently than control rats.

Figure 3 displays the feeding and drinking behaviour of rats microinjected with anti-/%endorphin antiserum (left panel) and anti-dynorphin A antibodies (right panel). Anti- /?-endorphin antibodies significantly reduced food and water intake when injected into the nucleus periventricularis hypothalami (hpv; P < 0.01). No significant change from their respective controls was observed after administration of the antiserum into the other areas of the VMH. However, combining the data of the areas of the VMH, where the respective controls are available (that is, hpv, hvmm, hdv, ar and mml), a significant reduction of food and water intake became apparent (P < 0.01). No difference was observed from the regions located outside the VMH.

A similar result was obtained with anti-dynorphin antibodies (right panel, Fig. 3). Individual comparison of the effect of antibodies of the areas of the VMH failed to reveal significant differences. However, when the data of these areas were combined and compared to the combined control data, there were significant reductions of food ( P < 0.05), and water intake ( P < 0.05). No significant differences were found for the data obtained from areas outside the VMH.

D i s c u s s i o n

The results reported here may add support to the concept that endogenous opioids tonically participate in the regula- tion of feeding behaviour. This conclusion is derived from

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Fig. 3. The effect of microinjections of anti-fl-endorphin antiserum (fi-End AB, left panel) and anti-dynorphin A antibodies (D YN AB, right panel) respectively, on food (�9 and water (O) intake in rats. For further explanations see Fig. 1. Additional abbreviations for the VMH: hvmc, nucleus ventromedialis, pars centralis;pd, nucleus premamillaris dorsalis; h vma, nucleus ventromedialis, pars anterior; F, columna fornicis; hp, nucleus posterior

experiments using antibodies to precipitate specific opioid peptides in the vicinity of their receptors.

The issue of which of the opioid peptides actually are responsible for the control of feeding has been pursued with antibodies directed against e-neoendorphin, fl-endorphin and dynorphin A1-13. A considerable inhibition of food and water intake was obtained with the anti-c~-neoendorphin antibody. Figure 4 schematically represents sections of the rat brain (K6nig and Klippel 1963), indicating the anatomi- cal structures microinjected with the antibody. Apparently, the VMH represents an area where the anti-c~-neoendorphin antibody elicited a response. And this area, although not exactly defined, is involved in feeding behaviour (Morgane 1961). The reduction of food and water intake (about 45%) is rather high considering the unilateral microinjec- tion. However, one has to bear in mind that administration of an antibody rather selectively neutralizes a specific opioid peptide and may, thus, have powerful effects. This rather selective action is in contrast with the wide spread of action of naloxone when given systemically (King et al. 1979).

Regarding a possible preference of the antibody to re- duce food or water intake our data indicate equi-effective action on both. Despite this result the possibility cannot be ruled out, for example, that the antibody affects primarily the water intake and that the effect on food reduction reflects the consequence of the primary action. A similar mechanism has been reported for the action of naloxone (Cooper 1980).

Less compelling are the results obtained with anti-E- endorphin antiserum and anti-dynorphin A antiserum. Microinjection of anti-/?-endorphin antiserum into the VMH largely failed to affect appetite when the individual

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Fig. 4. Sections of rat brain illustrating the anatomical structures microinjected with anti-c~-neoendorphin antibodies. The hatched areas represent structures within the VMH. Microinjection into these areas resulted in a significant reduction in food and water intake. The stippled areas are outside the VMH, and anti-c~- neoendorphin antibodies failed to affect appetite. The numbers refer to the section position according to the coordinates given by the atlas by K6nig and Klippel (1963)

areas are compared to the control injections. An exception was found for the periventricular nucleus of the hypothala- mus where the antiserum significantly reduced food and water intake. However, a combination of all the data obtained from injections into the VMH reveal a statistically significant effect of the antiserum on food and water intake. Thus, fl-endorphin may also be involved in the regulation of appetite. The weakest effects were obtained with anti- dynorphin A antibodies. Even a combination of all data from the injections into the VMH revealed only a very low effect on appetite, a borderline effect as judged by the degree of significance (P < 0.05). It should be noted that the iden- tical anti-~-endorphin antiserum and anti-dynorphin A antibodies proved effective in reducing the tonic inhibition by endogenous opioids controlling luteinizing hormone re- lease in prepubertal rats (Schulz et al. 1981).

These results draw attention to e-neoendorphin, which originates from pre-pro-dynorphin (Kakidani et al. 1982; H611t 1983). e-Neoendorphin has been shown to be pre- sented in high concentrations in the hypothalamus (Minamino et al. 1981 ; Weber et al. 1981 ; Maysinger et al. 1982). With respect to its opioid receptor selectivity it has been shown to combine with ~-receptors (Oka et al. 1982; Wfister et al. 1981b; Schulz et al. 1982) as well as with a-receptors (Schulz et al. 1982). Thus, the endogenous opioid involved in feeding behaviour in the VMH may be c~-neo- endorphin, rather than dynorphin A which is also an endogenous ~c-ligand (Wiister et al. 1981a; Chavkin et al. 1982). Therefore, the finding by Morley and Levine (1981)

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that exogenous dynorphin A given into the lateral ventricle causes feeding, is quite consistent with the present results. In addition, these data are not necessarily in conflict with the failure of the prototype ~c-ligand ethylketazocine to cause feeding (Tepperman and Hirst 1982; Sanger and McCarthy 1981), since the endogenous ~c-ligands, c~-neoendorphin and dynorphin A, behave only similarly, but by no means iden- tically, with ethylketazocine regarding their receptor interac- tion (Schulz et al. 1982). Finally, the question remains, why e-neoendorphin rather than dynorphin A controls appetite, although both opioid peptides show a parallel distribution in the brain (Maysinger et al. 1982; Weber et al. 1981). This parallelism does not imply that these peptides are identically processed or released since both can be manipulated (Millan et al. 1984) and processed differently (Seizinger, personal communication).

The possibility that/~-endorphin also regulates appetite should be considered. The data reported here may be in accord with the finding by Grandison and Guidotti (1977) that/~-endorphin injected into the VMH initiates feeding. They may even add to the recent debate regarding the signifi- cance of opioids and feeding (Gambert et al. 1980; Ettenberg et al. 1981; Przewtocki et al. 1983).

In conclusion, the present studies are suggestive for a function of c~-neoendorphin within the VMH in the control of food and water intake. However, these results do by no means exclude a critical function of other opioid peptides for feeding behaviour, even of those peptides to which antibodies have been microinjected. The various antibodies may display different kinetics, particularly with regard to penetration into the tissue and the synaptic cleft. Thus, the failure of an antibody to cause a specific effect in vivo does not necessarily imply a functional insignificance in ingestive behaviour of the peptide to which the antibody is directed.

Acknowledgements. We wish to thank Dr. V. H611t for providing some of the antisera employed. We are grateful to Dr. R. Mucha for helpful discussions and the stylistic revision of this paper.

References

Chavkin C, James IF, Goldstein A (1982) Dynorphin is a specific endogenous ligand of the kappa opioid receptor. Science 215:413-415

Cooper SJ (1980) Naloxone: effects on food and water consumption ,in the non-deprived and deprived rat. Psychopharmacology 71:1-6

Dupont A, Barden N, Cusan L, Merand Y, Labrie F, Vaudry H (1980) fl-Endorphin and met-enkephalins: their distribution, modulation by estrogens and haloperidol, and role in neuroendocrine control. Fed Proc 39:2544- 2550

Ettenberg A, Rogers J, Koob GF, Bloom FE (1981) Endogenous opiates and fasting. Science 213:1282

Ey PL, Prouse SJ, Jenkin CR (1978) Isolation of pure IgG1, IgGza and IgGzb immunoglobulins from mouse serum using protein A-Sepharose. Immunochemistry 15 : 429- 436

Gambert SR, Garthwaite TL, Pontzer CH, Hagen TC (1980) Fast- ing associated with decrease in hypothalamic /~-endorphin. Science 210:1271 - 1272

Goldstein A, Ghazarossian VE (1980) Immunoreactive dynorphin in pituitary and brain. Proc Natl Acad Sci 77:6207- 6210

Grandison L, Guidotti A (1977) Stimulation of food intake by muscimol and beta endorphin. Neuropharmaeology 16: 533-536

H611t V (1983) Multiple endogenous opioid peptides. Trends Neuro- sci 6:24--26

226

H611t V, Gramsch C, Herz A (1979) Immtmoassay of beta- endorphin. Radioimmunoassays of drugs and hormones. In: Albertini A, DaPrada M, Peskar BA (eds) Cardiovascular medicine. Elsevier, Amsterdam, pp 293- 307

H611t V, Haarmann I, Bovermann K, Jerlicz H, Herz A (1980) Dynorphin-related immunoreactive peptides in rat brain and pituitary. Neurosci Lett 18 : 149-153

Kakidani H, Furutani Y, Takahashi H, Noda M, Morimoto Y, H:irose T, Asai M, Inayama S, Nakanishi S, Numa S (1982) Cloning and sequence analysis of cDNA for porcine beta-neo- endorphin/dynorphin precursor. Nature 298 : 245 - 249

King BM, Castellanos FX, Kastin A J, Berzas MC, Mauk MD, Olson GA, Olson RD (1979) Naloxone-induced suppression of food intake in normal and hypothalamic obese rats. Pharmacol Biochem Behav 11 : 729- 732

K6nig JFR, Klippel RA (1963) The rat brain. Williams and Wilkins Company, Baltimore

Laschka E, Teschemacher H, Mehraein P, Herz A (1976) Sites of action of morphine involved in the development of physical dependence in rats. Psychopharmacologia 46:141 -147

Leander JD, Hynes III MD (1983) Opioid antagonists and drinking: evidence of re-receptor involvement. Eur J Pharmacol 87: 481-484

Locke KW, Brown DR, Holtzman SG (1982) Effects of opiate antagonists and putative mu- and kappa-agonists on milk intake in rat and squirrel monkey. Pharmacol Biochem Behav 17:1275-1279

Maysinger D, H611t V, Seizinger B, Mehraein P, Pasi A, Herz A (1982) Parallel distribution of immunoreactive e-neoendorphin and dynorphin in rat and human tissue. Neuropeptides 2: 211 - 225

McKay LD, Kenney N J, Edens NK, Williams RH, Woods SC (1981) Intracerebroventricular beta-endorphin increases food intake of rats. Life Sci 29 : 1429-1434

Millan MJ, Millan MH, Cztonkowski A, Herz A (1984) Contrasting interactions of the locus coeruleus as compared to the ventral noradrenergic bundle with CNS and pituitary pools of vasopressin , dynorphin and related opioid peptides in the rat, Brain Res 298:243 - 252

Minarriino N, Kitamura K, Hayashi Y, Kangawa K, Matsuo H (1981) Regional distribution of e-neoendorphin in rat brain and pituitary. Biochem Biophys Res Commun 102: 226- 234

Morgane PJ (1961) Electrophysiological studies of feeding and satiety centers in the rat. Am J Physiol 201:838-844

Morley JE (1980) The neuroendocrine control of appetite: the role of the endogenous opiates, cholecystokinin, TRH, gamma- amino-bytyric-acid and the diazepam receptor. Life Sci 27:3_5__5-_36.8 . . . . . . . . . _ .

Morley JE, Levine AS (1981) Dynorphin-(1-13) induces spon- taneous feeding in rats. Life Sci 29:1901 -1903

Morley JE, Levine AS, Grace M, Kniep J (1982) An investigation of the role of kappa opiate receptor agonists in the initiation of feeding. Life Sci 31:2617-2626

Oka T, Kazuro N, Kagiwara M, Watanabe Y, Ishizuka Y, Matsumiya T (1982) The choice of opiate receptor subtype by neo-endorphins. Eur J Pharmacol 79:301-305

Przewtocki R, Lasdn W, Konecka AM, Gramsch C, Herz A, Reid LD (1983) The opioid peptide dynorphin, circadian rhythms, and starvation. Science 219 : 71 - 73

Sanger DJ (1981) Endorphinerg_ic.mechanisms in the control of food and water intake. Appetite. J Intake Res 2 :193- 208

Sanger DJ, McCarthy PS (1981) Increased food and water intake produced in rats by opiate receptor agonists. Psychopharmaco- logy 74: 217 - 220

Schulz R, Wilhelm A, Pirke KM, Gramsch C, Herz A (1981) /~-Endorphin and dynorphin control serum luteinizing hormone levels in immature female rats. Nature 294: 757- 759

Schuiz R, Wfister M, Herz A (1982) Endogenous ligands for x-opiate receptors. Peptides 3 :973- 976

Tepperman FS, Hirst M (1982) Concerning the specificity of the hypothalamic opiate receptor responsible for food intake in the rat. Pharm Biochem B 17 : 1141 - 1144

Weber E, Roth KA, Barchas JD (1981) Colocalization of c~-neo- endorphin and dynorphin immunoreactivity in hypothalamic neurons. Biochem Biophys Res Commun 103:951-958

Wiister M, Schulz R, Herz A (1981 a) Multiple opiate receptors in peripheral tissue preparations. Biochem Pharm 30:1883 - 1887

Wiister M, Rubini P, Schulz R (1981 b) The preference of putative pro-enkephalins for different types of opiate receptors. Life Sci 29:1219-1227

Received September 23, 1983/Accepted March 26, 1984