Bidirectional control of palatable food consumption through a common benzodiazepine receptor: Theory and evidence

Brrrw Ke.secln~h BN//Pfi?l.

Vol.

15, pp. 397-410.

1985. ’ Ankho

Intematiord

Inc. Printed

0361-9230185

in the U.S.A.

$3.00

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Bidirectional Control of Palatable Food Consumption Through a Common Benzodiazepine Receptor: Theory and Evidence STEVENJ.COOPER Lkppurtmcnt of Psychology, Univrrsity of B~rmin~hurn, Birminghum

Bi5 2TT, U.K.

COOPER, S. J. B~~~~~~~.ti~j~~Ai contrd of p~~u~ubt~~~~~~ (.(J~lsu~~pfi~~~ f~zr~)z~~/? a wrnvw~~ heazodiazepiwreceptor: ?‘hectr> rendrrk&vw~~. BRAIN RES BULL. W(4) 397-410, 1985.-A classical approach to the control of food consumption has been to assume separate mechanisms for the arousal to eat. on the one hand. and the satiation of feeding responses, on the other. The present paper is concerned with a single, and a comparatively simple, neuronal mechanism which is endowed with properties to allow the complete determination of the levet of feeding, from hyperphagia to anorexia. The model for the control of feeding, which is presented here, draws attention to the benzodiazepine receptor found distributed through the brain, and present in certain hypothalamic nuclei. Recent evidence which characterizes the receptor is reviewed, and the various categories of benzodiazepine receptor Iigands are described. Pharmacological data, collected in a palatable food consumption model using non-fob-deponed rats, demonstrate that benz~iazepine receptor agonists produce hyperphagia, benzodiazepine receptor inverse agonists produce anorexia, and benzodiazepine receptor antagonists block both effects. Hence, bidirectional control of food intake can be achieved through differential ligand action at a common set of receptors. Speculatively, these data can be extended, if it is assumed that two endogenous ligands exist in the brain which act like benzodia~epine agonist and inverse agonist, respectively. Evidence for the presence in hypothaIamic nuclei of endogenous ligands of the latter kind is discussed. Benzodiazepine withdrawal-induced anorexia is atso described, and is taken as evidence for the part played by feeding mechanisms in the development of benzod~azepine physical dependence. Anorexia Appetite Benzodiazepines Benzodiazepine receptors Benzodiazepine Ct 218,872 CGS 8216 CGS 9896 DMCM FG 7142 Food consumption Phenobarbitone RoI5-1788 Tracazolate Zopiclone ____ -_-...____-

receptor ligands Palatability

subsequent thinking and experimentation especially with regard to feeding behaviour. Third, in a broad, imaginative sweep, each ‘centre’ was conceived as being under the control of multiple factors (sensory stimuli, internal environmental factors, extrahypothalamic brain structures, with recognition also given to the influence of learning). Fourth, Stellar presumed that these influences flowed into the hypothalamus, and that the outcome depended on the addition of all excitatory influences, with the subtraction of all inhibitory influences. The result determined the level of motivation to perform the response. Stellar’s article had little in the way of empirical evidenck to rely on, but was ambitiously synthetic, and looked forward to comprehensive investigations into the physiological bases of motivation. This project continues to be pursued vigorously today {92]. What could not have been anticipated at that time was the enormous growth in neuropharmacological and biochemical knowledge in relation to important questions about motivational control. Today, we are becoming accustomed to a rich complexity in the possible neurotransmitters and neuromodulators which are implicated in the control of feeding [Sl, 79, 801, and in the invoIvement of periphe~Ily-released peptides in the satiation of feeding re-

IN 1954, Stellar proposed a general account of the physiology of motivation [I 1I]. In his theoretical article, Stellar organized evidence which was available at the time into a urtifled framework, and laid out clear priorities for future research. Some features of his model are familiar, but it will make a valuable starting-point to the present discussion to be reminded of the main ideas which were incorporated into his system of motivation. First, Stellar was concerned with an account of motivated behaviour in general, and therefore drew on supportive evidence from studies of, for example, hunger, thirst, sex and activity. Second, he envisaged distinct neural mechanisms which were concerned with the excitation of motivated behaviour, and the inhibition of this excitation, respectively. These dual controls of motivated behavior were located principally in the hypothalamus. They were described as ‘centres’; the term “centre’ referring not so much to an anatomical entity, but more to some degree of functional localization. The important feature of the model was the spatial separation of mechanisms involved in the arousal of motivation, on the one hand, from those which produce the satiation of motivated behaviours, on the other. This ‘dual centre’ hypothesis held a particularly prominent place in

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sponses 121,461. It is natural, perhaps, to link the ‘dual centre’ idea with the intricate interactions of numerous neurotransmitter pathways to arrive at a complex model which accounts for the control of feeding responses [80]. Nevertheless, the aim of the present article is quite the opposite: it is to focus attention on a single and comparatively simple neuronal mechanism, and demonstrate that it is capable of mediating any level of feeding, from hyperphagia at one extreme to anorexia, at the other. The purpose is to show that the bidirectional control of feeding responses can in principle be achieved through actions at a single site, and to propose a scheme whereby the brain may utilise this site to regulate feeding. In addition, an explanation will be advanced to account for at least one form of pathological anorexia. The single mechanism in question was first identified as a drug receptor, and one which specifically binds the benzodiazepines selectively and with high affinity. The evidence which indicates that these receptors can control food consumption bidirectionally derives from recent pharmacological studies which will be reviewed here. The receptors are heterogenously distributed in the central nervous system, with dense concentrations occurring in several hypothalamic nuclei, including the ventromedial hypothalamic nucleus [ 1221. This novel model for the control of feeding responses, will best be explained if we proceed first with a description of the established and hypothetical elements which are involved, and then review the supporting pharmacological evidence, according to the following plan: (1) The pharmacological and biochemical characteristics of benzodiazepine receptors; (2) The actions of exogenous and endogenous ligands at benzodiazepine recognition sites; (3) An outline of the bidirectional control of feeding responses which are mediated by benzodiazepines receptors; (4) A description of recent pharmacological data which are consistent with the model; (5) Anorexia as a consequence of withdrawal following discontinuation of benzodiazepine treatments. BENZODIAZEPINE

1

3

FIG. 1. A hypothetical model of the GABA receptor-benzodiazepine receptor-chloride channel complex (view from outside the membrane above, and in section along the membrane axis below). The complex is formed by four monomeric peptide units, which are labeled l-4. These units are thought to contain at least three domains with different functions: C is the anion channel part, G the GABA-binding domain, and B the benzodiazepine-binding domain. The binding sites for channel agents, GABA receptor ligands and the three classes of benzodiazepine receptor ligands on the three domains are indicated on monomer 2. GABA receptor activation results in the opening of the anion channel, as shown on monomer 3. Ligands interacting with the C and B domains can ahosterically affect this anion gating process. Arrows on the fourth monomer refer to the multiple bidirectional interactions between the three domains. Reproduced with permission from [5l].

RECEFTORS

The benzodiazepines constitute a class of psychoactive drugs which are widely prescribed, and are clinically-useful because of their characteristic anxiety-alleviating, anticonvulsant, muscle-relaxant and hypnotic effects [47]. Pharmacological studies in animals have confirmed these effects exhaustively, and, in addition, have revealed effects of benzodiazepine treatments upon a wide range of behaviour, including operant performance [ 1011, feeding and drinking [ 19, 20, 241, memory [66,116], electrical brain stimulation [62], and social interaction [97]. It has been confirmed that a major effect of benzodiazepine treatments is to enhance GABA (y-amino butyric acid)-ergic neurotransmission throughout the brain and spinal cord, GABA being one of the most important CNS inhibitory neurotransmitters [31,52]. Lacking a direct effect on GABA receptors, benzodiazepines act in a modulatory sense to facilitate GABAergic transmission [30,49,53]. Following this link between benzodiazepines and GABA, an important step in discovering the molecular basis of their actions came with the identification of specific, saturable, and high-affinity binding to brain membrane sites using [“HI-diazepam as a radioligand [ 12, 13, 17, 76, 771. In virro and in viva studies indicated that the rank order of potency of benzodiazepines to displace a radioligand from membranebound benzodiazepine binding sites correlated highly with their clinical efficacy, and with their relative potencies in tests of muscle-relaxant, anticonvulsant, and anticonflict ef-

fects [13, 36, 64, 73, 76, 88, 89, 104, 1151. These results indicated that some proportion at least of the specific binding sites could be considered drug rrcrptors, in the sense of not only showing specific recognition of benzodiazepines, but also through their mediation of the biochemical and ultimately the behavioural consequences of benzodiazepine action. Regional studies revealed a heterogenous distribution of benzodiazepine binding sites in the central nervous system [13,17]. Using autoradiography, Young and Kuhar [122] showed that regions with high densities of sites included the molecular layer of the olfactory bulb, the mitral cell layer of the accessory olfactory bulb, the rostra1 median forebrain bundle, limbic system sites and various diencephalic areas. In the midbrain, high levels of benzodiazepine sites were detected in the superior colliculus and the substantia nigra, pars lateralis. In the cerebellum, the molecular layer exhibited very high levels of benzodiazepine sites. Using photoaffinity-labelling with [“HI-flunitrazepam, Mijhler and his colleagues [75] showed the locahzation of benzodiazepine binding sites in regions of synaptic contacts. By using antiserum to glutamate decarboxylase as a marker of GABAergic neurones, they demonstrated that one-third of the photolabeued benzodiazepine receptors were associated with immunocytochemically-stained nerve endings [78]. Re-

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cently, the visualization of benzodiazepine binding in the living baboon brain has been achieved using positron emission tomography [54], although the resolution is quite low. There is considerable evidence that the benzodiazepine receptor forms part of a larger membrane-bound structure which also includes the GABA* receptor and a chloride ion channel [51, 53, 87, 88, 108, 117, 118, 1191. Binding sites of the picrotoxinin- and barbiturate-type are also associated with this molecular complex [87, 117, 1181. With a voltageclamped cultured mouse spinal neurone preparation, Study and Barker [114] used the electrophysiological method of fluctuation analysis to investigate the mechanisms of GABAergic neurotransmission, and its modulation by diazepam (a benzodiazepine) and (-)-pentobarbitone (a barbiturate). Both benzodiazepines and barbiturates potentiate the increase in chloride ion flux across the neuronal membranes which underlies the inhibitory effect of GABA 167, 68, 711. They discovered that diazepam increased the frequency of channel openings with little effect on the average openchannel lifetime, whereas (-)-pentobarbitone decreased the frequency but increased open-channel lifetime. Both drugs increased the channel response to GABA, but did so by influencing channel kinetics in different ways. Figure 1 shows a schematic and hypothetical model of the receptor complex which incorporates three distinguishable functions, First, GABA recognition and binding occurs on the domain labelled G, from which control is exercised over the chloride channel C determining the frequency and average lifetime of the channel openings. Second, benzodiazepine recognition and binding occurs on the domain labelled B. Third, there is the chloride channel domain C which may also bind a variety of agents which affect anion translocation, i.e., channel-blocking convulsants (e.g., picrotoxin) and channel-facilitating and/or activating drugs (e.g., various barbiturates). The specific high-affinity binding sites for benzodiazepines are sensitive to agents which interact with GABA, receptors and chloride channels. Thus, benzodiazepine receptor agonists bind with greater affinity in the presence of GABA agonists and barbiturates [87, 117, 118, 1191. Benzodiazepines act at a site which is intimately coupled, physically and functionally, to GABA receptors and gated chloride channels. These characteristics have been confirmed in studies of solubilized and purified receptor proteins. Gavish and Snyder [421 concluded that after extensive purification procedures, the continuing association of GABA, benzodiazepine and chloride ion recognition sites suggested that they formed part of a single macromolecular complex. Using receptor protein solubilized with sodium deoxycholate and then purified, further evidence has been obtained that the GABA and benzodiazepine binding activities reside in a single macromolecular complex [ 107,112J. Sigel and Barnard [ 1061 have recently reported that the purified complex shows ligand binding not only to the GABA and benzodiazepine sites, but also exhibits high-affinity binding of TBPS ([“%I butyl bicyclophosphorothionate), a ligand for the chloride ion channel. Hence, a receptor complex can be isolated which retains specific binding sites for three different kinds of drugs: GABAA ligands, benzodiazepines and chloride channel-blocking agents. Interestingly, poly(A)mRNA which had been extracted from embryonic chick whole brain and microinjected into Xmopus oocytes directed the synthesis and membrane insertion of functional GABA-benzodiazepine-barbiturate receptor complexes [lOS]. Using an improved radiation inactivation method,

Chang and Barnard [ 181 estimated the molecular size of the benzodiazepine receptor in the synaptic membrane of brain cortex to be 220,000, and concluded that the same protein complex carried an associated class of GABA, sites. In addition to the discovery of benzodiazepine binding sites in the brain, sites have also been identified in peripheral tissue, including kidney, heart, pituitary, and adrenals [2, 5, 6, 171. The peripheral-type binding sites are pharmacologically distinguishable from the central-type, since [“HI RoS4864 or [:‘H] PK 11195 act as specific ligands for peripheraltype sites and show little affinity for central-type sites, whereas the converse is true for the potent benzodiazepine agonist, clonazepam [7,95]. Using [“HI Ro5-4864 binding, peripheral-type sites in the central nervous system have been detected, with a high density in the olfactory bulb, and a cellular and subcellular distribution which is quite dissimilar from that of the central-type sites [69,105]. There is evidence which suggests that peripheral-type sites may be associated with non-neuronal elements. However, there are also recent data that indicate that in the olfactory bulb, [“HI Ro5-4864 binding sites may be localized to olfactory nerves which originate in nasal epithelium [3,11]. BENZODIAZEPINE

RECEPTOR

LIGANDS

Radioligand binding techniques enabled relationships between benzodiazepine binding affinity and pharmacological activities to be explored, and also led to the development of non-benzodiazepine compounds which nevertheless show high-affinity binding to the same sites. As indicated above, good correlations have been obtained between clinical efficacy (and characteristic benzodiazepine effects in animals) and the affinity of benzodiazepine compounds for their recognition sites. Williams [119] identifies nine classes of non-benzodiazepine anxiolytics, some of which bind with high affinity to benzodiazepine sites. Zopiclone [9] and suriclone [ 101 are both cyclopyrrolone derivatives which bind with high affinity to central benzodiazepine sites. CL 218,872 is a triazolopyridazine [63,1 lo] with anxiolytic activity. Benzodiazepine binding sites in the cerebellum, designated Type 1, exhibit high affinity for CL 218,872, in contrast to a second type of benzodiazepine receptor (Type 2) which is found in hippocampus [81]. The concept of central-type benzodiazepine receptor heterogeneity has been the subject of much recent discussion [45, 60, 65, 70, 95, 1191. CL 218,872 binding to the benzodiazepine receptor is enhanced in the presence of GABA [81], and is sensitive to photoaffinity labelling [74]. Unlike benzodiazepines, however, CL 218,872 binding to benzodiazepine sites is insensitive to stimulation by chloride ions, etazolate and pentobarbitone [81]. It is possible, therefore, that triazolopyridazines may interact with the benzodiazepine-GABA receptor complex in a way which is different from that of the benzodiazepines. CGS 9896 is a pyrazoloquinoline with high affinity for benzodiazepine recognition sites and the binding is enhanced by GABA [43,44]. While CGS 9896 possesses anxiolytic properties, it appears to be relatively devoid of sedative or musclerelaxant effects [8,109]. The pyrazolopyridines, etazolate, cartazolate and tracazolate, show anxiolytic activity in rat conflict test procedures [4, 72, 1191. The mechanism of action of the pyrazolopyridines is unknown, but interestingly they enhance both benzodiazepine and GABA binding in rat brain membranes [72,119]. Data on some of these non-

Gln-Ala-TLr-yal-Gty-Asp-Yal-Asn-TLrAsp.Arg.Pro-Gly-Leu

- lsu.Asp-leu.Lys

FIG. 3. Amino acid sequence of an octadecaneuropeptide obtained by trypsin digestion of DBI (diazepam binding inhibitor) and which is a ligand of benzodiazepine recognition sites (see [X31).

FIG. 2. Structures of so”,, P-carboline-3-carboxylates: JKCM (methyl ester), /3-CCE (ethyl ester). /3-CCPr (n-propyl ester), FG 7142 (N’-methyl-~-Caroline-3-carboxamide), DMCM (methyl 6,7Reproduced with dimethyl-4-ethyl-P-carboline-3-carboxylae). permission from [SO].

benzodi~epiue ~nxiolytics will be described later, in connection with palatability-induced feeding in non-fooddeprived rats. A major pharmacological advance in the study of benzodiazepines saw the intr~uction of specific benzodiazepine receptor antagonists, with high aS%nity for benzodiazepine recognition sites and little or no intrinsic activity. The imidazobenzod~~epine derivatives, Ro 15 1788 and Rot53505, effectively antagonize behavioural, electrophysiological and biochemical effects of benzodiazepines [50,53]. Ro15-1788 binds with high affinity to central-type, but not to periphe~-type, benzodi~epine receptors; it does not differentiate between Type 1 and 2 binding sites in the brain. In many instances, although not in ail, it appears to be devoid of agonistic activity 150,531. CGS 8216 is a phenyipyrazoloquinoline derivative /121], structurally related to CGS 9896. It shows high affmity for specific brain benzodiazepine sites, and functions as a potent benzodiazepine antagonist [34]. In behavioural studies, it does exhibit some activity when administered in large doses [23,24, 39,401 but not invariably so [24, 37, 961. Further pha~a~ologi~al developments, however, introduced a third class of agents which bind to benzodiazepine receptors (inverse agonists, contragonists, or agonists with negative efftcacy), and led to proposals for a three-state model of the benzodiazepine receptor [ 15, 16, 56, 57, 84,91, 1191. In the fist instance, ethyl p-carboline-3-carboxylate (P-CCE) was obtained from human urine, and was shown to have high affinity for benzodiazepine receptors 1141. Not only was /XCE found to antagonize effects of benzodiazepines, but was also found to possess intrinsic actions which were detected as a proconvulsant effect [32, 83, 861, and as an anxiety-producing effect 182,911. Within the &carboline series (Fig. 2), the methyl and propylesters were established as convulsant and anticonvulsant, respectively. The most potent convulsant p-carboline has proved to be DMCM (methyl 6,7-dimethoxy-4-ethyl-@arboline-3-carboxylate) [85,90]. FG 7142 is a stabilized @-carboline, which

is proconvulsant 1991, is claimed to induce anxiety in human volunteers [35], and appears to be anxiogenic in some animal models of anxiety [41,91], although not in all f33]. Since some members of the /?-carboline series (Fig. 2) bind with high affinity to benzodiazepine receptors, display agonistic activity, yet produce effects which are contrary to those of classical benzodiazepines, they have been described as inverse agonists, or by some related term. Currently, ligands for benzodiazepine sites are thought to span a spectrum of activity from full agonist, partial agonist, antagonist, partial inverse agonist, to full inverse agonist [ 15, 16,83.91]. Partial inverse agonists include /3-CCE and FG 7142, while DMCM is an example of a full inverse agonist [573. Pole ct al. [94] proposed a ‘three-state model’ of the benzodiazepine receptor in which benzodiazepines act as agonists to enhance the function of the benzodiazepine receptor as a coupling unit between the GABA receptor and the chloride channel (Fig. I). fi-Carbolines, in contrast, act as inverse agonists to reduce this coupling function. Antagonists like Ro15-1788 are thought to block the effect of both agonists and inverse agonists on GABAergic synaptic transmission. Recently, the existence of benzodiazepine receptor mixed agonis~antagonists have been proposed, a category which is said to include CL 218,872 and CGS 9896 [ 120).

The discovery of specific, high-affinity benzodiazepine receptors in the brain immediately suggested the possible existence of a natural endogenous l&and for ligands) for the receptor. Recently, Guidotti and colleagues [48] extracted and purified a polypeptide from rat brain, which was shown to be a competitive inhibitor for the binding of I”H] diazepam, [:‘H] flunitrazepam, p-[“H] carboline ethylester, and :‘H-labelled RolS-1788. The peptide was designated DBI ~diazep~ binding inhibitor), and contains 105 amino acid residues, with a relative abundance of basic.residues, particularly lysine. DBI, like p-carbolines, abolished the increase of [“H] GABA binding which was induced by diazepam. This similarity between DBI and p-carboliue activity was confirmed in behavioural experiments using a shock-suppression of licking test. Previously it had been shown that p-carbolines (FG 7142, P-CCM, DMCM, p-CCE) enhanced the suppression of licking produced by a low intensity shock (0.35 mA) delivered whilst thirsty animals licked at a drinking spout (termed a ‘proconflict’ effect) 1281.In the same test, DBI also exhibited a proconflict conflict with a shock level set at 0.25 mA, and this effect was reversed by the benzodiazepine receptor antagonist, Ro15-1788 [28]. Cyanogeu bromide cleavage of DBI produced three peptide fragments, but only the N terminal fragment (F,) retained the behavioural activity of DBI [29]. DBI purified from rat brain homogenates was treated with trypsin, to yield seven peptide fragments ]381. Only one of these fragments showed an ability to inhibit [3H]-diazepam binding and pos-

FIG. 4. DBI (diazepam binding inhibitor) immunostaining in telencephalic regions (A-E) and in cerebellum (F-G). Panel A: Low magnification (X30) ofa coronal section of the rat brain showing high densities of DBI-positive cells in medial amygdaloid nucleus (am), arcuate nucleus (see Panel C), posterior nucleus of the hypothalamus (ph), dorsal premamillary nucleus (pm), and in the paraventricular nucleus (pv) of the thalamus. Panel B: DBI-positive cells and fibres in the CA3 region of the hippocampus, localized in the basket cell(b) layer. (x880). Panel C: High density of DBI-positive cells and fibres in the arcuate nucleus of the hypotha~mus. t x 700). Panel D: A single DBi-positive neurone with a long axon in the cortical amygdaloid nucleus. (X800). Panel E: Highest density of DBI-positive cells and fibres in the cortex is localized in the layers III-IV. (x 130). Panel F: Low magnification of the rat cerebellum showing dense innervation of the molecular layer (m) and the DBI-containing neurones in the granular cell (g) layer. (x30). Panel G: High magnification of the rat cerebellum showing DBI-containing Golgi neurones (arrows) in the granule layer. Purkinje cells (p) are unstained but they are densely-innervated and surrounded by small DBI-positive cells (arrowheads). (x880). Reproduced by courtesy of Dr. H. Alho.

D

COOPER tivity, 350?24 pmol/mg prot). followed by the ventromedial (190-e 14 pmoVmg prot), supraoptic (1702 14 pmol/mg prot), and other hypothalamic nuclei, the periaqueductal grey and the cerebellular cortex. Much lower concentrations were found in nucleus accumbens, caudate-putamen and cortical regions (60?8.0-4459.0 pmolimg prot). Dense networks of immunoreactive neurones were detected in many hypothalamic nuclei (Fig. 4A and C), in the amygdala (Fig. 4A and D), in the hippocampus (Fig. 4A and B). and cerebellum (Fig. 4F and G). It seems probable that DBI is the precursor of a family of peptides, which include natural endogenous ligands for benzodiazepine recognition sites and which have ‘inverseagonist’-like activity. Interestingly, the highest concentrations of DBI-like immunoreactivity are found in hypothalamic nuclei of the rat brain, a brain region long known to be associated with feeding behaviour and the regulation of body weight [60, 111, 1131. These important, new findings identify natural ligands for brain benzodiazepine receptors, and indicate that their activity may be comparable to that of the inverse agonist P-carbolines (Fig. 2). It should be noted that there are also a number of other naturally-occurring compounds which bind to benzodiazepine sites [ 1201. While their status as physiologically-relevant ligands has yet to be determined, one cannot rule out the possibility that endogenous substances may bind at benzodiazepine sites to produce diazepam-like activity. WI11

mral

SllOS

SlOCl

FIG. 5. Model for the bidirectional control of palatable food consumption in non-food-deprived rats as a consequence of the sequential release of two hypothetical endogenous ligands which occupy a single set of benzodiazepine receptors and induce differential receptor protein conformations. Stage 1: Pre-feeding state, with feedingcontrol benzodiazepine receptors unoccupied (open circles). Feeding is initiated with the release of a hypothetical feeding-promoting peptide (FPP-solid circles). Stage 2: Feeding begins and is maintained when the receptors are occupied by FPP and a conformational change is effected (solid circle in box). As feeding proceeds, a second endogenous ligand is released, hypothetically

designated as a satiety-promoting peptide (SPP-dotted circles). Stage 3: When this second ligand occupies the benzodiazepine receptors in the set, the receptors adopt a new conformation (dotted circles in hexagon), the physiological effect of which is to promote the cessation of feeding. Stage 4: The feeding bout or meal is terminated when excess benzodiazepine receptors are occupied by SPP. The novel feature of this model is that a single receptor system can mediate enhancement or suppression of feeding responses, depending upon the nature of the receptor protein conformation induced by ligands.

sessed a proconflict action in the punished licking test. This biologically-active fragment obtained by trypsin digestion of DBI proved to be octadecaneuropeptide (ODN), with an amino acid sequence which is shown in Fig. 3. The authors suggest that DBI is the precursor of ODN, and that there may be at least two ODN replicas in DBI [38]. Their evidence cannot, as yet, exclude the possibility that natural effector of the benzodiazepine recognition sites could be smaller than ODN. Using immunohistochemical and radioimmunoassay methods, the selective neuronal distribution of DBI has now been described for rat brain [l] (see Fig. 4). The highest content of DBI-like immunoreactivity was found in the arcuate nucleus of the hypothalamus (DBI-like immunoreac-

BENZODIAZEPINE-RECEFTOR MEDIATED BIDIRECTIONAL CONTROL OF FEEDING: A MODEL

The fundamental assumption of this model of feeding behaviour is that there are benzodiazepine receptors in the brain, which can undergo ligand-controlled conformational transformations, the consequences of which are modulated changes in food consumption varying from hyperphagia to anorexia. The model has been partially validated pharmacologically with respect to palatability-induced feeding responses in the rat; the system may function separately from food deprivation-induced feeding (procedural details and relevant data are provided in the next section). Figure 5 illustrates the type of feeding control which is envisaged. In addition to the set of feeding-related benzodiazepine receptors, two hypothetical neuropeptide ligands for the receptors have been included as elements in the model. The initial stages of feeding are associated with the selective release of the first neuropeptide, which binds to the benzodiazepine receptors, inducing them to adopt an ‘agonist’ conformation. This change in conformation interacts with other components of the receptor complex (Fig. 1) to affect the gating of the chloride channel. In consequence, feeding is enhanced. As feeding proceeds, however, a second neuropeptide is released, which also binds to the benzodiazepine receptors, the effect of which is to induce a different conformational state (one associated with inverse agonists). This second neuropeptide, may be derived from the DBI precursor, for example ODN, identified by Costa’s group (Fig. 3). The two hypothetical neuropeptides are assumed to bind competitively to the same population of benzodiazepine receptors. Later. the first neuropeptide is no longer released, and there is exclusive release of the second neuropeptide, which binds to the receptors to cause the termination of feeding. Progress through the meal, therefore, is associated with an allosteric change in the benzodiazepine receptors from an ‘agonist’-binding conformation to an ‘inverse agonist’-binding conformation. Hence the motivation

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FIG. 6. Dose-response functions for the hyperphagic effect of five henzodiazepine receptor agonists and of phenobarbital in male nonfood-deprived rats. The results show the meantS.E.M. for the amount fg) of palatable food consumption which occurred under conditions of complete f~iliarity in a 30 min test period. N= IO. per group in all cases. Within each drug condition, separate groups of animals were used for each dose condition and for the corresponding (V) group. In,jections were administered IP, 25-30 min prior to the feeding test. Levels of significance for comparisons of individual groups against control: *p
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to feed is postulated to reflect, in quite a direct sense, the conformational states operating throughout the population of relevant benzodiazepine receptors. Pharmacologically, this model predicts hyperphagic effects of bentodiazepine receptor agonists, anoretic effects of benzodiazepine receptor inverse agonists, antagonism of both effects by receptor antagonists, and a mutual cancellation of effect when benzodiazepine receptor agonists and inverse agonists are co-administered. The evidence in support of the bidirectional control of feeding is reviewed in the next section. BENZODIAZEPINE-RECEPTOR MEDIATED BIDIRECTIONAL CONTROL OF FEEDING: DATA

The majority of the data which will be reviewed refer to a palatable food consumption paradigm which we recently adopted [ZS]. Briefly, freely-feeding male rats were familiarized over a period of days to handling, injection pro-

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FIG. 9. Chlordiazepoxide (CDP, 5.0 mg/kg IP) produced a significant hyperphagic effect in a 30 min test of palatable food consumption in non-food-deprived male rats. This effect was completely blocked by CGS 8216 (M-20.0 m&kg IP). CGS 8216 given alone produced significant reductions in food intake at 10.0 and 20.0 mg/kg IP. Results are shown as mean?S.E.M. (N=lO per group). Levels of significance in comparison with the control group (0): *p
cedure, and to a highly palatable diet. The recipe for the diet was to mix 50 ml NestE’s sweetened condensed milk, 200 ml tap water and 150 ml (100 g) finely-milled rat food (prepared fresh before each feeding test). Animals were individuallytested in cages identical to the home cages. The diet proved to be extremely attractive to the rats, who consumed 14-17 g of the food within a 30 min test period. The latency to begin feeding was negiigible, and the rats appeared to be highly aroused in anticipation of food presentation. The motivation to feed depended on the palatability of the diet, and not on food-deprivation or any other disturbance of the animals’ normal physiological state. A range of benzodiazepines were found to stimulate the cons~ption of the palatable diet in a 30 min test (Fig. 6). Mean levels of food consumption reached 25 g, requiring the animals to eat throughout much of the test period. From Fig. 6, it can be seen that there were potency-differences between the drugs which were tested: a rank-ordering would be clonazepam, lorazepam > diazepam, midazolam > chlordiazepoxide. This order is consistent with the relative potencies of these drugs to inhibit [“HI diazepam or [SH] flunitrazepam binding 113, 36, 64, 761, and therefore indicates an effect which is mediated by actions at benzodiazepine receptors. Figure 6 also shows that phenobarbitone, which does not bind to benzodiazepine receptors but which may act at another site on the receptor complex controlling chloride channel opening (Fig. l), exerted a strong hyperphagic effect, producing a mean consumption of nearly 30 g in the test. When rats were pre-fed the diet prior to drug administration to produce partial satiation (Fig. 7), the feeding response was strongly reinstated by injection of midazolam, a short-

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FIG. 10. The p-carbolines DMCM and FG 7142, benzodiazepine receptor ligands which act as inverse agonists, and CCS 8216 produced dose-related reductions in palatable food consumption by non-food-deprived male rats in a 30 min test. IP administration. Levels of significance in comparison with corresponding (V) control groups: *p
acting benzodiazepine agonist [93]. Partial satiation of the feeding response, therefore, did not appear to inhibit the hyperphagic effect of the benzodiazepine treatment. Midazolam did not have any longer-term hy~rphagic action beyond the first 30 min of the test (Fig. 7B). Zopiclone, a cyclopyrrolone derivative, which binds with high affinity to benzodiazepine sites, also produced a doserelated hyperphagic effect (Fig. 8). A hyperphagic effect of zopiclone has also been reported recently in a different type of feeding paradigm 11021. The t~azolopyridazine, CL 218,872 also produced hy~~ha~a, but the dose-response relationship appeared to be biphasic (Fig. 8). Significant increases in food consumption were detected at 2.5 and 5.0 mg/kg IP, but not when larger doses were administered. Not all putative anxiolytics produced hyperphagia, however. Following tracazolate administration, the only change to occur was a depression in palatable food consumption at higher doses (20.0 and 40.0 mg/kg IP), associated with general behaviour depression. Likewise, the non-sedating anxiolytic CGS 9896 [8], administered over the dose range 2.5-20.0 (IP and PO), had no hyperphagic effect [25]. Using a different feeding paradigm, Sanger ct ul. [102] reported small nondose related increases in food intake during a 2 hr test period, fo~owing CGS 9896. The hyperphagic effect of benzodiazepines can be antagonized by the benzodiazepine receptor antagonists RolS1788 [25] and CGS 8216 (Fig. 9). Since centrally-acting benzodiazepine receptor agonists exert strong hyperphagic effects, whereas the peripheral-type receptor ligand RoJ-4864 has no hyperphagic effect in the palatable food consumption test [25], it can be concluded that the hype~hagia is mediated by actions at central benzodiazepine receptors. RoS-3663 is an atypical benzodiazepine, which electrophysiologically behaves as a GABA antagonist [ 1031, and which binds to the picrotoxinin site associated with the chloride channel (Fig. 1). It would be expected to have an anorectic action, and so it does 1251. Interestingly, clonazepam at a dose which produced a marked hyperphagia completely blocked the anoretic action of RoS-3663 [25]. This result suggests functionally-significant interactions between the benzodiazepine receptor domain and the chloride channel domain (Fig. 1).

BENZODIAZEPINE

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405

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FIG. 11. The anorectic effect of FG 7142 (20.0 mg/kg IP) on palatable food consumption in a 30 min test was reversed by benzodiazepine receptor antagonist Rol5-1788 (2.5 and 5.0 mg/kg IP). Results show meantS.E.M. (N=lO per group). Levels of significance in comparison with FG 7142 alone group (0): *p
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FIG. 12. The anorectic effect of FG 7142 at 5.0 mg/kg IP (upper panel) and 10.0 mg/kg IP (lower panel) was reversed by the benzodiazepine receptor antagonist CGS 8216 (1.25-5.0 mg/kg IP). Results show mean?S.E.M. (N=8 per group). Levels of significance in comparison with the control group (Cl): *[r
0

FIG. 13. The hyperphagic effect of clonazepam the anorectic effect of FG 7142 (10.0 mg/kg IP) cellation. When administered in combination, sumption in the 30 min test was virtually the level (N=9 per group). **p
(1.25 mg/kg IP) and showed mutual canpalatable food consame as the control test), in comparison

@Carbolines which act as inverse agonists at benzodiazepine receptors produced a profound dose-related anorexia in the palatable food consumption test (Fig. 10). The effect was also shown by CGS 8216 when given in doses which exceed those at which it acts as an antagonist with no intrinsic activity (c.f. Fig. 9). The anorectic effect of FG 7142 was antagonized by the benzodiazepine receptor antagonists Rol5-1788 (Fig. 11) and CGS 8216 (Fig. 12). It was not re-

versed by a period of 24 hr food deprivation prior to the feeding test [22]. The data of Fig. 13 illustrate that benzodiazepine receptor occupancy can be associated with hyperphagia (clonazepam alone), no change from baseline (clonazepam + FG 7142), or anorexia (FG 7142 alone), respectively. Thus, bidirectional control of palatable food consumption followed from the conjoint actions of two compounds with agonistic activity, but which differed in terms of positive and negative efficacy [ 151. The relationship between these data and the mode1 proposed in Fig. 5 is clear; two neuropeptides acting at the same benzodiazepine sites could in principle produce hyperphagia or anorexia, depending on their relative concentrations. The similarity between the behavioural activity of one naturallyoccurring neuropeptide which binds to benzodiazepine receptors, ODN, and that of the P-carbolines (including FG 7142) has already been noted [38]. Thus, it would be interesting to examine the effects of ODN on palatable food consumption, and to determine its interactions with both benzodiazepine agonists and antagonists. The results reported in this section are summarized in Table 1. BENZODIAZEPINE WITHDRAWAL-INDUCED ANOREXIA Electrophysiological,

behavioural

and clinical data indicate

406

~‘OOWR TABLE

I

FUTUREPROSPECTS

SUMMARY OF THE EFFECTS OF BENZODIAZEPINE RECEFI-OR (BR) AGONISTS AND INVERSE AGONISTS, ALONE AND IN COMBINATION WITH OTHER FACTORS ON THE CONSUMPTION OFAPALATABLEDIETIN NON-FOOD-DEPRIVEDRATS BR agonist e.g.,

clonazepam

BR inverse agonist e.g.. FG 7142

(a) alone

(b) combined with Ro15-1788 CGS 8216 FG 7142

hyperphagia

anorexia

antagonism antagonism mutual antagonism

antagonism antagonism

BR agonist RoS-3663 food-satiation food-deprivation ?=not

no change no change 3

mutual antagonism ‘, ‘, no change

tested.

that withdrawal symptoms which follow the discontinuation of chronic benzodiazepine treatments are opposite in nature to the characteristic benzodiazepine effects [58,98, lOO]. For example, anxiety, irritability, shakiness and sleeplessness commonly affect individuals in the immediate period following gradual withdrawal of benzodiazepine treatment [58]. In addition, a significant proportion of patients who were studied in a double-blind, placebo-controlled study showed loss of appetite, and disturbances of taste and smell after stopping the medication [%I. Anorexia has also been reported to occur in animals following discontinuation of benzodiazepine treatment [59,98]. The anorexia and perceptual disturbances which occur as part of the symptoms of benzodiazepine withdrawal may be secondary to the anxiety, sleep disturbance and physical discomfort which is present. Alternatively, the loss of appetite in withdrawal may indicate that there was a direct action of the benzodiazepines during medication at brain receptors which are involved in the control of feeding responses. A loss of the motivation to feed as part of withdrawal, may indicate that physical dependence on benzodiazepines relates in part to changes in feeding-related mechanisms. A possible mechanism to account for benzodiazepine withdrawal-induced anorexia is that discontinuation of medication in the physically-dependent individual results in an increased activity of an endogenous ligand, which acts as an ‘inverse agonist’ at benzodiazepine receptors. The characteristics of feeding behaviour in the context of drugwithdrawal in physically-dependent subjects is a neglected research area, and hypothetical mechanisms to account for the changes in appetite should serve a useful function in stimulating additional experimentation.

A considerable amount of evidence confirms that benzodiazepine treatments produce overconsumption of food in many animal species [ 19,241. The hyperphagic effect in the present series of experiments on palatable food consumption was mediated by actions at benzodiazepine sites, since potency differences amongst several benzodiazepines correlated well with their afftnities for benzodiazepine recognition sites, and the effect was reversible by benzodiazepine receptor antagonists. The new departure in the present data is that drug action at benzodiazepine receptors can also induce an anorectic effect. when ligands are used which have been characterized as inverse agonists. In principle, therefore, a single set of receptors could mediate opposite effects on feeding behaviour, depending on allosteric conformational changes which are induced by various ligands. Two endogenous ligands, one acting like a benzodiazepine agonist and the other acting like an inverse agonist, would be sufficient to control any feeding response from hyperphagia to anorexia through a single set of benzodiazepine receptors. Three important questions now arise. First, where are the putative feeding-related benzodiazepine receptors located‘? The high concentration of DBI-immunoreactivity within hypothalamic nuclei (Fig. 4), and the long-standing association between hypothalamic nuclei and the neural control of feeding responses, suggests that efforts should be devoted to exploring drug and peptides actions at benzodiazepine receptors in these nuclei. Second. what are the endogenous ligands for benzodiazepine receptors involved in feeding motivation? The model for the control of feeding responses which is proposed assumes that at least two exist, one which promotes feeding responses and one which inhibits them, through complementary actions at the same set of benzodiazepine receptors. ODN (Fig. 3), or some related peptide derived from DBI, may function as a satiety neuropeptide; a prediction which is open to experimental test. The existence of a second endogenous ligand released to initiate and maintain feeding responses is speculative at present. Further research on endogenous ligands should reveal whether or not there are multiple endogenous ligands for benzodiazepine receptors involved in feeding. Third, more information is needed on the factors which may modify benzodiazepine-receptor control of feeding responses. Neither food-deprivation nor feeding satiety appear to be importantly involved (Table I), at least so far as feeding responses to a highly palatable diet are concerned. Future research might profitably focus on possible interactions between oropharyngeal factors. and the effects of benzodiazepine agonists and inverse agonists on the level of food consumption.

ACKNOWLEDGEMENTS I wish to thank Dr. H. Alho and Prof. W. Haefely for providing photographs, Prof. E. B. Barnard and Dr. E. Costa for sending reprints. D. Barber provided invaluable technical assistance. Mrs. Barbara Hudson expertly prepared the manuscript.

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BENZODIAZEPINE

BIDIRECTIONAL

CONTROL

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\ t~r,r-

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1981.

BENZODIAZEPINE

79. Morley,

BIDIRECTIONAL

CONTROL

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