A game with shifting mirrors

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473 cause @-CCB accumulation either by inhibition of its release, or by release of a benzodiazepine agonist.

A game with shifting mirrors Borges phrase,

invented the felicitous ‘U game with shifting mirrors’, and coyly attributed it to an apocryphal Indian writer, Mir Bahadur Ali. The phrase could well be applied to the study of neurotransmitter regulation of anxiety and learning. What was true yesterday is doubtful today; what was unbelievable yesterday is likely today; anxiety and leaming and stress may or may not be reflected by the same mirror(s), and the mirror(s) has shifted or is shifting.

fwhhrbolines and benzodiazepine receptors

Eirst came the P-carbolines. In I980, Braestrup and his colleagues isolated n-ethyl-fi-carboline-3carboxylate from brain and urine extracts’. It was immediately apparent that the rather drastic use of a methanol-HCl mixture at 8O’C for 2 h, caused the ethyl ester group to be introduced during the extraction procedure*#*. However, it was also clear that the p-carboline nucleus might have originated in the brain tissue*+, with the possibility that other fi-carboline esters could be formed in the brain. Ethyl-P-carboline-3-carboxylate and a variety of synthetic analogs, including its amide (FG1742), a methyl ester (P-CCM), and methyl-6,7-dimethoxy-4-ethyl-bcarboline-3-carboxylate (DMCM) were found to bind to central-type benzodiazepine receptors with very high affinity, to be displaced from those receptors by benzodiaze&es, and to exert psychopharmacological actions generally opposite to those of benzodiazepines (inverse agonism)‘i These inverse agonist actions included the induction of severe anxiety in humans (for example, by FG-1742; Ref. 3), a decrease in the time spent by rats in the open arms of an elevated plus maze (a measure of increased anxiety)-, a facilitation of certain types of learning6, and a pro-convulsant or outright convulsant effect (see Refs 2 and 6). All these effects, like those of agonist benzodiazepines, could be blocked by the specif;c competitive antagonist flumazenil

(Ro 15-1788) presumably by displacing the fi-carboline esters from receptor binding sites (see Refs 5, 6 and 20). Recently, De Robertis and his co-workers extracted n-butyl-P;zcif;;;tca;z$ate @CCB) ‘I. Unlike the previo’usly extracted p-carboline esters’, @CCB is unlikely to be an extraction artifact: it was extracted with water, at neutral ‘pH and in the cold’,“. It was purified by several reverse-phase HPLC systems and identified as p-CCB by UV absorption, HPLC and mass spectrometry. It has a Kd of - 3 nM and high specificity for benzodiazepine receptors9,10. p-CCM acts as an inverse agonist at central-type benzodiazepine receptors, being proconvulsant’* and displaying anxiogenic activity, as measured in two psychopharmacological tests; this effect was antagonized, in mice, by a very low dose of flumazenil. Brain p-CCB levels doubled after exposure of rats to acute swimming stress, and this change was counteracted by the prior injection of diazepam13. These data suggested that p-CCB could be the endogenous benzodiazeene receptor ligand involved in the generation of anxiety that everybody had been looking for12*13. However, the failure of the previously described P-carboline esters to live up to this title demands caution. Although P-CCB, unlike the other esters, appears to be a true endogenous extractable in an compound aqueous phase9*“, it has yet to be detected in a synaptic vesicle fraction and shown to be released from this fraction by stress; moreover, until shown otherwise, an increased brain level could well be indicative of retention rather than release. That something that binds to central-type benzodiazepine receptors is released by stress is suggested by the finding that maximum [3H]flunitrazepam binding is diminished after exposure of rats to acute swimming plus elevated stress or an maze14*15. But in an attempt to cope with stress the brain could

Brain benzodiazepioes And suddenly, brain benzod&epines came onto the scene. De Blas and his co-workers in Stony Brook purified N-de,+ methyldiazepam, a metabolite of diazepam, from aqueous extracts of bovine and rat brain by immunoaffinity chromatography using a monoclonal antibody to the benzodiazepine 3-hemisuccinyloxyclonazepam16*17. The substance isolated from brain was identified as N-desmethjrldiazepam in binding studies and by mass spectrometry, reversephase HPLC analysis, and W spectrophotometry16. A compound with the same absortlum spectrum and I-IPLC profile as oxazepam was also isolated16**7. The same group detected benzodiazepine immunoreactivity in human and rat brain, including human brains kept in paraffin since 1940, sixteen years before benzodiazepines were first synthesized by the indust@ (Fig. I). These findings were confirmed by two other groups who used chemicd extraction procedures different from those used by De Blas: conventional purification; and extraction methods not employing immunoaffinity. Wildmann et al. in BasellRI and De Robertis and co-workers in Buenos Aires”*‘*, extracted substances that they identified as benzodiazepines from bovine and rat brain. The De Robertis group identified diazepam from brain extracts by its profile in several reverse-phase HPLC systems, by its LJV absorption spectrum, and by its recognition by the monoclonal antibody to diazepam employed by De Blas. Perhaps most important, Medina et al.” found that the molecule they identified as diazepam was concentrated in synaptic vesicles, and to a lesser extent in synaptosome cytosol, of bovine brain gray matter. In addition, the molecule identified as probably being diazepam in rat adrenal medulla” has also been found in the plasm.: including species, of several humans”, and in cow’s milk”. While it is perhaps premature to be categorical that there is diaz-

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Y

epam in the brain. the data are highly suggestive. Indeed, Ndesmethyldiazepam and oxazepam are well known metabolites of diazepam; and it is hard to believe that different groups, using different extraction and analytical procedures11~‘5*18~19,inspectrometry15, cluding mass should find molecules in the brain that are not distinguishable from these substances and all be wrong. The finding that a molecule like diazepam, or diazepam itself, is contained mostly in synaptic vesicles” suggests that it could serve as a neurotransmitter or neuromodulator. This is supported by the fact that the benzodiazepine receptor antagonist, flumazenil, has a variety of psychopharmacological effects of its own, generally opposite to those of the benzodiazepines2’. At high doses, its effects may be construed as being anxiogenic in laboratory animals: it increases the !atency to eat in a novel environmer@ and, most importantly, facilitates some

Fg. 1. Bindingof anti-benzodiazepine monoclonalantibodiesfo human cerebellum that had been storad in paraffin since 1940 in the absence (A) and presanca (B) of diazepam. +, Purkinje cell layer. (Taken, with permission, from Ref. 16.)

forms of leaming22-24. However, flumazenil is also anticonvulsantz5, can be anxiolytic in some behavioral test@, and is generally without effect in the elevated plus maze test?‘; these results are certainly atypical of a true inverse agonist. While the possibility that flumazenil may have an intrinsic activity of its own20J5 cannot be ruled out, the evidence suggesting that brain @CCB and brain diazepam may be real transmitters opens up interesting possibilities. File and Pellowzo, and Medina et al.” have suggested that a balance between endogenous benzodiazepine agonist and inverse agonist mechanisms may play a role in the regulation of the perception of, or the response to, anxiety or stress. Recent findings suggest that this may be the case for the regulation of learning, at least in circumstances of stress or anxiety. The effects of flumazenil on leaming (or reversing the effects of injected benzodiazepines or

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fi-CCB) stand out because they are seen at much lower doses than those needed to observe any other psychopharmacological effects. Low, non-anxiogenic doses of flumazenil (5.0 mg kg-’ or less), given prior to training, enhance retention of habituation to a loud, startling buzzera3,24, and active= and inhibitory avoidance leaming23224 in rats; but not habituation to the much less stressful simple exposure to an open field23:24. This suggests that acquisition of the three former tasks may normally be downregulated by an endogenous mechanism involving benzodiazepine agonists, and is in line with the findings that mild forms of acute stress, such as swimmingI or exposure to the elevated plus maze15, decrease benzodiazepine receptor binding in the brain. The decreased binding suggests that there may be a release of endogenous benzodiazepine receptor ligands in the brain in those circumstances; and behavioral training procedures are often mildly stressfuP6. The effect of flumazenil on acquisition suggests that a benzcdiazepine agonist (diazepam, perhaps?) could be released by the stress or the anxiety that accompanies some f0rrr.j of leaming23,24. All of which raises many questions. l Assuming j3-CCB and diazepam really exist in the brain, where do they come from? The f_3-carboline

structure could be produced in the brain by condensation of aldewith indolealkylamines hydes and/or tryptophan. Indeed, harman (2-methyl-g-carboline) and tetrahydro-P-carboline have long been known to exist in brain tissue (see Ref. 10). However, direct evidence for the endogenous synthesis of @CCB or any other like substance is lacking2. Diazepam and desmethyldiazepam have been found in potatoes, rice, maize, lentils, soybeans and other plants used as food’8*27, and in cow’s milk”, so their presence in the brain may derive from an o~gin16.27_ alimentary Microorganisms, including some that may contaminate food, synthesize benzodiazepines2’. The three groups who found benzodiazepines in brain work under the general assumption that they are mainly of alimentary originlO,l ,16,18,

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1989 [Vol. 201

but do not disregard the possibility that at least part of the molecules could be synthesized in the brain. De Blas and his co-workers have reported the presence of benzodiazepine immunoreactivity in the neuroblastoma x glioma hybrid cell line NG 108-15 grown for three months in a serum- (and benzodiazepine-) free medium17. So far, however, the hypothesis of an alimentary origin of brain benzodiazepines seems the most parsimonious. Many, of course, would turn up their noses at the mere thought of a putative transmitter that is directly imported from food to brain in its final form. However, it should be borne in mind that all neurotransmitters come from food: the brain amines and the peptides derive from amino acids; the choline of acetylcholine comes with lecithin; and so on. In some cases (noradrenaline, the peptides), several enzymatic steps are needed in order to transform the compounds from food into transmitters; in others (5-HT, histamine), just one or two steps will do it. Diazepam could be a case in which no steps are needed in order to transform what is eaten into a fully fledged transmitter. l

Is there any reason to think that

brain diazepam can function as a transmitter? The reasons are

several and compelling, but not decisive. Unless one assumes that the great variety of physical, chemical and immunological procedures used by De Blas in the USA, by Wildmann et al. in Switzerland and by De Robertis et al. in Argentina all went wrong and gave misleading data, it seems reasonable to think that, yes, there is diazepam in the brain, and, yes, it is concentrated in synaptic vesicles”. What would a substance known to bind to definite subsynaptic receptors be doing in synaptic vesicles, to begin with, if not getting ready for mischief? And then, there are the findings with low doses of flumazenil on its own, particularly its effects on learning during stress; and the findings of receptor occu ancy changes caused by stress 14,P5. Certainly, this is suggestive, but it is not enough. Studies of localized changes in synaptic vesicle diazepam levels

475 after stress and/or learning, and synaptosomal diazepam uptake and release experiments will be needed before one can swear that brain diazepam is a transmitter. @ If both p-CCB and diazepam are transmitters, what is their role in anxiety, stress and/or learning? pCCB could generate anxiety12,13, like other fi-carbolines had been proposed to do4; or it could be retained by cells during anxiety so as to avoid making things worse; or diazepam could be released by stress or anxiety, so as to overcome their effects, or in order to inhibit fi-CCB release. Or there could be a balance between endogenous @CCB- and diazepammediated mechanisms that could shift the perception of, or the reaction to, stress or anxiety one way or the other (see Refs 11 and 20). Or, as often happens in multiple choice tests and life in general, none of the above.

Do /WCS, diazepam and flumazenil help us to understand the relationship, if any, between regulation of anxiety and stress, and learning? The relationship l

between stress (or anxiety) and learning is becoming clearer. A bit of arousal, or of anxiety or even stress, is perhaps necessary for learning, and too much hinders learning (see Refs 26 and 29). This process was long thought to be regulated mostly or exclusively by stress hormones and brain catecholamines acting in the posttraining period26. The recent findings on the effect of fi-carbolines on leaming6*23,30, particularly the effect of p-CCB23, and on the effect of flumazenil on the leaming of (specifically) anxiogenic tasks22-24, suggest that acquisition during anxiety or stress may be downregulated by release of endogenous benzodiazepine ligands. Cl

q

q

Which brings us back to Borges’ ‘a game with shifting phrase, and/or mirrors’. First, anxiety that just stress were things happened. Then, they were treated with benzodiazepines. Then P-carboline esters came along and were thought to be the generators of anxiety. Then they fell into disrepute because they were shown to be extraction artifacts. Then (J-CCB appeared; it

was shown not to be an artifact, but people are cautious about whether or not it is a physiologically active substance. On the agonist side of the mirrors, first nobody knew, then most did not believe, and now we all know that there may be endogenous benzodiazepines in the brain. They do not appear to be artifacts, and they are contained in synaptic vesicles. The question remains, however, of whether or not they are there for some purpose. Three years ago nobody would think, and now there are some reasons to think, that brain benzodiazepines may be real neurotransmitters. They could be involved in coping with stress, and/or in the downregulation of learning during stress. A game with shifting mirrors indeed, in which all of us who eat potatoes or rice or soybeans or maize or drink milk 0,11.1&19,27 may be inadvertent players. (The phrase, ‘it must be something I ate’, is not by Borges.) The shifts are so rapid, however, that most workers in the field have become increasingly careful with their interpretations. Perhaps because a broken mirror brings seven years of bad luck. IVAN IZQLJIERDO Centro de Memoria, Departamento de Bioquimica, batituto de Biociencins, UFRGS (centrot, 90049 Porte Alegre, RS, Brazil.

References 1 Braestrup. C., Nielsen, M. and Olsen, C. (1980) Proc. Nat/ Acad. Sci. USA 77, 2288-2292 2 Braeshup, C. (1988) Ncurochrv~. ht. 13, 21-24 . 3 Dorow, R., Horowski. R., Pascelke, G., Amin, M. and Braestrup, C. (1983) Lancet ii, 98 4 Prado de Carvalho, L. et nl. (1983) Adv. Bioclrenl. Psyclzopl~amraco-ol. 38, 175-187 5 File, S. E., Pellow, S. and Braestnrp, C. (1985) Pharmacool. Bioc$r,tz. B&W. 22. 941-944 6 File, S. E. and Pellow, S. (1988) Bebav. Brain. Rrs. 30,31-36 7 Braestrup, C., Schmiechen, R., Neef, G., Nielsen, M. and Petersen, E. N. (1982) Scie?rcc216, 1241-1243 8 Jensen, L. H., Stephens, D. N., Sarter. M. and Petersen, E. N. (1987) Brain Res. Bull. 19. 359-364 9 Pena, C., Medina, J. H., Novas, M. L.. Paladini, A. C. and De Robertis, E. (1986) Proc. Nat/ Acad. Sri. USA 83. 4952-4956

10 De Robertis, E., Pena, C., Paladini, A. C. and Medina, J. f-t. (1988) Nruroclrmr. ht. 13. 1-11 11 Medina, J. H., Pena, C., Piva, M.. Paladini, A. C. and De Robertis, E.

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12 Novas. M. L.. Wolfman. C., Medina, 1. H. and De Robertis, E. (1988) Phanvocol. Biochenr. Behov. 30, 331-336 13 Medina, J. H., Pena, C., Novas, M. L.. Paladini. A. C. and De Robertis, E. (1987) Neerorhem. hit. 11, 255259 14 Medina. J. H., Novas, M. L. and De Robertis, E. (1983) Eur. 1. Pharmoccl. 96, 181-184 15 Rage. L., Kiivet, R. A., Harro, J. and Pold. M. (1988) Naunyn-Schmiedeberg’s Arch. Pharmncol. 337.675-678 16 Sangameswaran, L:, Fales, H. M., Friedrich, P. and De Blas, A. L. (1986) Proc. Nat! Acad. Sci. USA 83, 9236-9240

It has become textbook knowledge that the major inhibitory neurotransmitter receptor, the GABA* receptor, is a multisubunit receptor-channel complex which can be allosterically modulated by two important classes of drug, the berzodiazepines and the barbiturates. The primary structures of the GABA* (Ref. 1) and glycine’ receptors were defined in 1987, showing that these two receptors were members of a ligand-gated ion channel receptor superfamily. Since then, there have been major efforts to determine the number, stoichiometry and function of the GABA* receptor subunits. However, instead of clarifying further the ‘classic’ pharmacological picture, this recent molecular biological onslaught has revealed unexpected complexities. Early work3 using affinity purified GABAA receptor preparations suggested the receptor complex to be composed of only two subunits, the 50-53 kDa a: subunit and the 55-57 kDa B subunit. However, recent discoveries by DNA homology cloning of novel receptor subunits (y, 6 and E) (Refs 4 and 5; P. H. Seeburg et al., unpublished) and subtypes of subunits (ul-~4, Bl-B3, yl, y2 and others) (Refs 6-9, P. H. Seeburg et al., unpublished) provides many combinatorial possibilities for the formation of functional receptors. How near has this taken us towards defining GABA* receptor in-vivo stoichiometry, and understanding modulatory mechanisms at the molecular level? Reconstitution studies with the cloned ti and B subunits have not yielded receptors that inimic in-vivo

17 De Bias, A. L., Park, D. and Friedrich, P. (1987) Brain Res. 413, 275-284 18 Wildmann, J. et a!. (1987) J. Nertral Tramnsnz. 70, 383-388 and 19 Wildmann, J., Niemann, J. Matthaei, H. (1986) 1. Neural Trawn. 66, 151-160 20 File, S. and Peilow, S. (1986) Psychoph0n?lac02ogy 88, l-11 21 Bodnoff, S., Suranyi-Cadotte, 8. E., Quirion, R. and Meaney, M. J. (1989) Behav. Neurosci. 103,20%212 22 Lal, H., Kumar, B. and Forster, J. M. (1988) FASEB J. 2,2707-2711 23 Pereira, M. E., Medina, J. H. and Izquierdo, 1. Braz. I. Med. Biol. Res. (in press)

some indeed, pharmacology; studies have demonstrated agonist responses elicited by benzodiazepine antagonists and inverse agonists! However, recent work has demonstrated that coexpression of 01and j3 subunits with the y2 subunit yields a functional receptor which can be modulated in a predictable way by benzodiazepines. A combination of such expression and localization studies is starting to unravel the functions of individual GABAA receptor subunits. Homomeric channel formation Electrophysiological and/or pharmacological analyses of expressed receptor subunits in either Xenopus oocytes” or cultured mammalian cells” have shown that functional homomeric, or single subunit, receptors can form. All GABA* receptor subunits examined to date form homomeric channels5Jo*1* each of which is activated by GABA and potentiated by barbiturates such as pentobarbital. These results are surprising since photoaffinity labelling studies had earlier indicated that GABA analogues such as 13Hlmuscimol bound the Bsubunit only’2-‘4; however, they are consistent with suggestions that the Cl- channel itself is the site of action of the barbiturates. Homomeric channels are relatively inefficient in that only small whole cell currents are observed”*” (and high concentrations of GABA are required for gating,. see below). These currents increase in magnitude as additional subunits are included in the expression experiments. Homo-

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24 Izquierdo, f., Pereira, M. E. and Medina, J. H. Behav. Neural Biol. (in press) 25 Jensen, E. H., Petersen, E. N. and Braestrup, C. (1983) Life Sci. 33,393-399 26 McCaugh, J. L. (1989) knnu. Rev. Newosci. 12, 255-287 27 Unseld, E., Krishna, D. R., Fischer, C. and Klotz, U. (1988) Trends Neurosci. 11,

490 28 Luckner, M. (1984) Secondary Metnbolism in Microorganisms, Plants and Animals (2nd edn), Springer-Verlag 29 Izquierdo, I. (1989) Behav. Neural Biol. 51, 171-202 30 Venault, P. et al. (1986) 864-866

Nature 321,

merit channel formation has also been demonstrated for the neurnicotinic acetylcholine onal receptor15 and the strychnine binding (48 kDa) subunit of the glycine receptor16*17. In the latter case, relatively efficient channel formation was observed. Since homomeric GABA* receptors require higher concentrations of GABA for channel gating5,“*“, and none of the subunits show differential GABA activation, it is likely that more than one receptor subunit plays a role in channel activation. _k-vivo receptors show a positive cooperativity of GABA activation (Hill coefficients >l.O) which may reflect the ability of several different subunits to respond to the neurotransmitter. To date, expression of cloned receptor subunits has not revealed this property, although positive cooperativity has been seen with expressed glytine receptors16. Single channel patch clamp analysis of homomeric ~1, a2, a3 or Bl GABA* receptor subunits also reveals that many of the single channel properties of these receptors are qualitatively the same as or similar to those seen in normal heterooligomeric receptors”. Multiple subconductance states, with preferred conductances of 19 pS and 28 pS for all three or-subunits and 18 pS and 27 pS for the B-subunit have been obseived. These values compare favourably with the GABA& receptor subconductance states seen in spinal cord neuronsls. Benzodiazepine binding sites Despite evidence that the (Ysubunit forms the benzodiazepine binding site14,19, detailed analyses of the benzodiazepine responsiveness of both human” and