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GABA* receptormodulationof memory:the role of endogenousbenzodiazepines

TiPS-july

1991 [Vol. 121

presence in neuroblastoma or glioma cell cultures’) suggests that they may be endogenously synthesized (see Refs 7, 9, 10, 12).

Early evidence for GABAA mechanismsinmeInory Systemic picrotoxin administration has long been known to Ivan lzquierdo and Jorge H. Medina enhance memory retentioP; indeed, this was the first evidence that GABA* mechanisms could be GAB& receptors are known to downregulate memory consolidation proinvolved in memory storage”. crsses: picrotoxin and bicuculline enhance memory, and benzodiazepines and However, this went unheeded at mlr.w$nol depress it. 7’he discouery of naturally occurring benwdiazepines in the time, because the inftuence of the brain prompted a recent inoestigation of whether these compounds could picrotoxin on GABAergic bansact as physiological regulators of the GABA* receptors inoolved in memory mission was not recognized until molulotion. Different forms of learning cause a rapid reduction of several years later. The first paper benzodiarepine-like immunoreactivify in septum, amygdala and hippoto suggest that GABAergic mechcampus; microinjection of the benzodiarepine antagonist ffumazenil into these anisms could downregulate conregions, at the time f!rat consolidation is taking place, enhances memory. Ivan solidation was published in 197% lzquierdo and Jorge Medina suggest that these and other findings indicate and referred to the deleterious fhot benzodiazepines released in the septum, amygdala and hippocampus do effect of the daily post-training indeed physiologically downregulate memory storage processes; moreouer, administration of the GABA benzodiazeaine release could be modulated by the anxiety and/or stress associated ‘with each type of lewning. transaminase inhibitor aminooxyacetic acid on retention of an active avoidance behaviouP. The including rats and Memories are labile immedispecies, enhancing effect of picrotoxin on humans”” (see Fig. l), mainly ately after acquisition and remain located in synaptic vesicleslO.Brain memory was mcentltl17confirmed decreasingly so for a period of a inavarietyoftasks benzodiazepines may be of alifew minutes, during which they The amygdala is rich in GABA* mentary origin, since they are are susceptible to both deleterious and GABA,, mceptom and plays found in numerous plants that and facilitatory influences that serve as food and also in milk9*‘0, a role in memory storage pfodetermine how much of each cessC?P’. Recent findings suggest although some evidence (e.g. their be memory will eventually that GABAergic mechanisms in storecP2. The initial lability of the amygdala regulate memory memories is considered to be consolidation. Re&ntion of inhibieither the cause or the effect of tory avoidance in rats is reduced a process of consolidation, the intensity of which can be modulated bythepost-kainingWWI@ala by neurohumoral and hormonal injection of the GABA* receptor mechanisms’. Treatments given agonist muscimol, and is enhan& shortly before or after training may by injection of the GABA* affect consolidation; the latter, receptor antagonist bicucuUinc’“. being devoid of influences on acin Thus GABA,, mechanisms quisition, are usually preferred’*‘. the amygdala may downregulate Most of the mechanisms that memory consolidation. In admodulate consolidation are facilidition, post-tmining intraamygdala tatory’,‘. Recent studies indicate microinjection of the GABAs that there are several GABA sysreceptor antagonist back&n imtems, mediated by GABA&receppairs memory*9, suggesting that tor complexes, that downregulate GABAs xecepto~, which block the memory and are modulated by release of a variety of neurotransbrain benzodiazepine receptor mitten?, may play a similar role. IigandP. Benzodiazepine molSome data indicate that GABAecules have recently been deergic synapses in the septum scribed in the brain of several may also be invohred either in the downregulation of recently acquired memories, or in acqui1. ftquicrdo is Professorof Ncurochcmisfry sition itself. PrC-training intrawd Head of the Cm&o dc Memorio. ~cporrseptal muscimol inje&on hinders omtnto de Bioquimico. Instituro dr Bio&ncins, retention of ’ tasks in rats Uniwtid~dc Fcdmt do Rio Cmndc do Sul IUFRGS) W49 Port0 Alqe. RS. B&I, and (a radial maz t3- or a Morristank=). I. ff. Medin is Awci& Profr5.w of Physio&y md Herd of tht Laborotorio de Ncrro~rept~rrs. bMMo de hoto@ Cclulrrr, Focultod de Medidnr. Uniocrsidad dr Burros Aim. Poroguoy 2155.3cr piso. 1121 Buenos Aim. Argentina.

Effectof systemic fhunazenil on Benzodiazepines have long been known to impair many

TiPS- /lriy 1991[Vo!. 121 forms of learning in many species, including humans (see Ref. 23). In view of the recent discovery of benzodiazepines in brain”“, and of the evidence that brain GABA* mechanisms could be involved in the regulation of memory storage processes, several groups investi. . .. .. . . . . gatea me possioiuty that brain benzodiazepines play a part in this regulation (see Ref. 12). The first experiments involved systemic injections of the benzodiazepine receptor antagonist flumazenil (R0151788)ssJ~*~. At a low, non-anxiogenic dose (5 mg kg” i.p.), flumazenil, given before training, was found to enhance retention of habituation to a buzzersJs, and of acti~e?~ and inhibitory (passive) avoidances*zs in rats. The effect of flumazenil on active avoidance was also seen -.-I__ _..~_L L.-l-~- J_--_ _* .Lumng mucn rugner aoses or me drug (l&N mg kg(s-‘)2’. Pre-training administration of a variety of inverse agonists at benzadiazepine receptors also enhanced retention of aversive and other behaviours in rats26*w(and see Refs 9, 12). The naturally oc(.&&g9J0$B inverse agonist butyl &carboline-3-carboxylate @-CCB) also enhanced the retention of habituation to a buzzer and of inhibitory avoidance at doses 5-20 times lower than those reported to be anxiogenic or pro__-___l___&Js ‘inc pL_err= _U__&“I _t 0p-LLD FI”P Lun”-r--~. on memory is antagonized by a low dose of flumazenil (2 mg kg-‘), ineffective on its own; this same low dose of flumazenil aiso antagonizes the pre-training amnntic effect of diazepam and clonazepan?. (The antagonism by flumazenll of the amnestic effects of diazepam was first described in humans several years aSoN.) l%unazenil affects retention only of fairly stressful or anxiogenie behaviours such as various forms of avoidancesJ425, and l..&&&..S&~.. mI. ..I_ &Us o; ;o ;i ‘ I(IYI,WI.“LII*r .” II “U&&S, restricted environments”. It does not affect the retention of less stressful tasks, such as habituation to an open fields~2s.This is in apparent contrast to the old finding that picrotoxin, another blocker of CABA,, transmission, enhances memory of nonstressful behaviour such as maze learning in rats13. This possibly results from the different sites and modes of action of the two drugs. Flumazenil acts upon a modu-

261 latory receptor and merely attenuates sensitivity to CABA,, agonis&; picrotoxin, by its action on the Cl- channel, bl& CABAergic transmission altogether. A comprehensive study comparing the effects of flumazenil and picrotoxin (or bicuculline) on a wide ninge of iearriing and memory tasks is needed to dissect these differences. Plumazenii has both benzodiazepine-like and fi-carbolinelike behavioural effects, which a few years ago was taken to indicate that it may possess intrinsic agonist or inverse agonist activity”. Today, we are more IikeIy to attribute’* the behavioural effects of flumazenil to antagonism of one of the recently found naturally occurring benzodiazepines or f&l-CCB, or the various -_-.Z>_ It-__>_ _I ___.-_I pepnue uganas or cenrrar L____ oenzudiazepine receptors described previously by Costa’s groups. The effects of fhunazenil on leaming suggest that training releases endogenous ligands of an agonist na@*‘*; and brain benzodiazepines contained in synaptic vesicles’o are obvious candidates,

(As will be seen below, brain benzodiazepines are, indeed, released by training.) Post-training flumazenip, l3CCB’” or benzodiazepines4*5*33.M dre usually, but not invariablp35, ineffective on memory processes. This led to speculation that these substances acqui;~~ . . rather than ~?~datnm*~ Systemically administered benzo: diazepines, however, reach peak blood or brain concentration Slowly: in 20-30 min or more (see Ref. 36) and i.p. flumazenil takes 10 min to reach peak blood levels (I. Izquierdo, unpublished). l’herefore, benzodiazepine ligands may indeed influence consolidation but if administered systemically after training they reach the brain too late, when consolidation has occurred. As discussed below, _____L >-I. _L..%_.> recent oara oucameo using intracerebral fhunazenil injections4”fo support this view.

The findiigs discussed above suggest that benzodiazepines might be released in the brain by

Pr&aMng~~.Tmatmentsgivenbefmethetminingsessienin which there is acquisition (i.e. before a hrn@ vxpeihm);

in principk, such lfeabtmts may affect both rcquiaition and col¶soEdatim.

~.TreatMltrigi~afeWsWXlda~mimltUirRa pacquisition (i.e. after a training session or learning ex+ence). They are believed to act mainly or exclusivelyon the consolidation process. A~~.Lceming~a~tdorcseh+wapsinfitlotothenvirc~ abk sttmufusby mng (activepooidaner) or mfmtningfnnn p&rming (Wbitory or pas&e dvoidsnce)some spactfk rrspolu+. lnbibttory avoidu\acm~k~iredinancbiel(i.e.Inafmscconde)mdis therefore very widely used tn memgr experiments. Habitudion.Leamingtoinhlbitorientin formof learning.

?-iPS-/u/y 1991IVOI.I.21

262

training experiences. This was investigated further by measuring immunobenzodiazepine-like reactivity in different brain regions of control rats and rats submitted to two different forms of training in the same apparatus (a 50 x 25 x 25 cm box)‘.“. Immunoreactivity, quantified by radioimmunoassay using a monoclonal benzodiagainst antibody azepines’,“, was measured in rat cerebral cortex’*” amygdala4s6, septum, hippocampus and cerebellumb. First, large regional differences were found in control animal$. Benzodiazepine immunoreactivity levels were highest in the medial septum, followed by the amygdala, hippocampus, cerebral cortex and cerebellum. Regional differences in benzodiazepine immunoreactivity were compatible with the distribution of GA6AA receptors in the brain (see Refs 3,9). Secondly, the silnple free exploration of the training apparatus during a one-minute period - a procedure that causes habituation to that environmentb - was followed by a decrease of benzodiazepine-like immunoreactivity in the cortex (38%), amygdala (28%) and medial septum (55%), but not in the hippocampus or cerebellum. Inhibitory avoidance training in the same box (animals received a footshock as soon as they stepped down from a start Iatform and began explorin~4*6 % ) was followed by a much more pronounced decrease of benzodiazepine-like immunoreactivity in the cortex (62%), amygdala (76%), medial septum (92%) and hippocampus (83%), but not in the cerebelIum6. The depletion data could be explained by a release of benzodiazepines from synaptic vesidesC followed by rapid diffusion’! Because the depletions occurred within seconds and the catabolism of benzodiazepines takes hours36, one cannot explain the depletion of benzodiazepines resulting from habituation or avoidance training by a sudden enhancement of the catabolism of these substance@, and thus vesicular release is the best explanation. A component of the response to habituation training and that to inhibitory avoidance training must be identical because in both

cases the animal receives the same initial stimulus: exposure to the box. However, the additional footshock stimulus given in the avoidance training inhibitory results in a greater release of benzodiazepines in ai1 structures measured4nb. Iniracerebralinjections of flumazenil and memory Immediate post-training bilateral injection of fhimazenil (5 nmol each side) into the medial septum and amygdala enhanced retention of the inhibitory avoidance task, but not of habituation; intrahippocampal flumazenil administration enhanced retention of the two tasks4sb.This could be attributed to antagonism of the benzodiazepines released during and/or after training in these structures. The effect of fLiazenil on avoidance learning correlates well with findings of a pronounced release of benzodiazepines in medial septum, hippocampus, and amygdala after training in this taskb. GABAergic transmission in the amygdala and septum is suspected to, or does, inhibit various neuronal systems involved in consolidation (cholinergic, NMDA and others3J2-“). Intraseptal or intraamygdala flumazenil may have no effect on habituation6 because the apparent release of benzodiazepines in these structures was lower after habituation than aher avoidance training. By contrast, while intrahippocampal flumazenil did affect retention of habituation, this task was not followed by a detectable depletion of benzodiazepines. This suggests that a tonic, rather than a phasic, release of benzodiazepines in the hippocampus plays a mle in the post-training processing of habituatior?. There are two types of tonic electrical events in the hippocampus that have been imp!icated in post-training memory storage processes: theta rhythm%, and long-term potentiation (LTP)“. both are under the inhibitory control of GABAA synapse&+0 and would thus be expected to be affec:ed by flumazcnil, which hinders benzodiazepine-modulated GAUAergic transmission’ (SW below). The GABAergic control of both theta rhythm” and LTPQois complex, and involves at

least two different sets of synapses in each case: those between basket and pyramidal cells, and others between extrinsic neurons and pyramidal cells. Mechanism of action of flumazenil If the benzodiazepines released in the amygdala, septum and hippocampus are involved in memory modulation by GABA systems, flumazenil would be expected to reduce sensitivity to the amnestic effect of muscimol. This was studied in the amygdala using an inhibitory avoidance task. As discussed above, the immediate post-training intraamygdala injection of fhunazenil causes memory facilitation of this task4sbM. Post-training intraamygdala administration of the GABAA agonist nu+,drnol (O.oOSO.5nmol) caused retrograde amnesia415. &-training flumazenil (2.0 or 5.0 mg kg-’ i.p.) reduced the sensitivity of the amygdala to the effect of post-training muschnol by a factor of at least 100 (Ref. 4). This is consistent with the argument that benzodiazepines reIeased during and after training sensitize amygdala GABA,, receptors and that flumazenil blocks this sensitization3*4*6ss.It also agrees with the finding that the amygd& becomes 100 times more sensitive to the amnestic effect of muscimol during trainings. In summary, these findings support the hypothesis that posttraining memory consolidation processes are normally downregulated by a GABAT mechanism in the amygdah’ , and also demonstrate that the mechanism is of the GABA* type, that it is modulated by brain benzodiazepine agonists, and that flumazenil facilitates memory by influencing these mechanisms. The findings on benzodiazepine reiease, and on the effect of flumazenil microinjections in the septum and hippocampus, strongly suggest that the memorymodulating benzodiazepineGABA* systems in these structures operate in the same way as in the amygdala6sJD. Blockers of GABA-associated Clchannel5 Ro54864 is both an a onist at peripheral-type benz d tszepine

TiPS - july 1997 /Vol. 121

Efiect ofanxiety

263

levels

on

by the A mechanisms

Learning exmences are accompanied by varying levels of alertness, anxiety or stress depending on the type of experience or tasW. The amygdaia, septum and hippocampus recognize and respond to alertness, ~_. 1-L. __I,_ _..~___ .I., amenng I.** ._ activation of anxlery anwor suess *_...1.. 1eve18Ynm various neu ro&ansmitter systems”. These systems, in hwn, modulate memory consolidation processes,which take place either in those same structures or at their projection s&P. Once ConsoMated, memories become stable and are not susceotible to such modulath.

The amsolidation

anxiety

+i

process I& for a few seconds

or minutes after each learning experience*(see Fig. 1).

experiences u--l

actuation of

at GAGA, receptor complex

I

1

-_Cyz benzociiazepine 63 release in:

C

GABAA, re&pior activation

d

4 opening of CT channel

i

ai

-

d

0a-

The specific effecta of snxiety on memory consolidation am shown in Fig. 2. Anxiety &aws be-i-

azepines in the septum, amygdala snd hi-pus (Fit. 2, aP. These shtanm est upon central-type bcnzodiuapiae secqhxs from which they csn be displaced by ffumszenil (Rol!Wgg) (Fig. 2, b); the systemiC+~intrasqtaf, fntrasmygdala or intmhippocampal aamin&ation offI_ enhances memory consolidation. The effect is different deoendlna on the is-inject& addance leaming is e&mced by flumazenil given into any of the three iIhuctures, whereas spatial b&u&m lsarninp is enhim& only by intrship&unazenifs . Benzodiazepines enhance the binding of GABA or muscimol to GABAAreceptom (Fig. 2, c); oost-training muscimol administration

ta muscfmol (ai feast in the amygdafa~. Activation df the CABA* receptors opens an intrinsic Cl- channel (Fig. 2, d); blockers of this channel (R&g64 or picrotoxin, administered systemically or intracerebrally) enhance memory consolidation7b.

receptors, and a blocker of the GABAA-related Cl- channels; in the channel, it binds at a site close to, but not identical to, the picro-

RECEPTOFt COWLElI

-

CI0

Reftra~ts 1 lzqukrdo,I. (1969)&kav.Neurel Bid.51,17l-X? 2 McGqh, 1. L (1989) BmirrRn. Bull. l3.339-315 3 32, L. R. (1987) Memory and Iwit, Oxford Univenity

4 Izquierdo, I., Per&a, M. E. and Me&a, J. H. (199OlB&m. 5 6 7 8

Nwmf Riot.54, W-41 D4 Cunha, C. d 8l. Bmz. /. Med. Biol. Res. (in Press) lquhdo, 1. et rl. (1990) kbm. Nmrl W. 54.1054~ Woumm, c. et al. (1991) Bmin Res. 546,74-80 Da Cunha, C. et al. (1991) Brain Rrs. 544,03-M

toxin site, and that can be antagonized by the isoquinoline carboxamide, PK11195*1J2. The immediate post-training

administration of R054M4 (1-5 mg kg-’ i.p.; 2.5 wg per rat, i.c.v.; 1.640 ng, intraamy dala) caused memory facilitation” f . he-training

rips - ]lIh/ 1997/Vol. 121

2b-l

i.p. administration of this drug has no effects. Both the posttraining effect and the lack of pre-training effect of Ro54864 are reminiscent of picrotoxin’3. A dose-response curve was found for intraamygdala microinjections of RoW864, which was antagonized by PK11195, and shared by picrotoxin”. As expected, the effect of picrotoxin was not antagonized by PK1119543, which like tio54%4 binds to a site in the Cl- channel different from that of picrotoxin”.“. These findings further endorse the hypothesis that there is a mechanism in the amygditla that downregulates physiologically memory and involves benzodiazepine-GABAA receptor complexes, because post-training blockade of the GABA*-related Cl- channel, using compounds that block the channel at two ditterent sites, enhances memory. Anxiety, stress and learning The benzodiazepine-GABA,, mechanisms in the septum and amygdala downregulate the retention of avoidance; in the hippocampus, these mechanisms also downregulate the memory of habituation to a novel environment. It is reasonable to suppose that a rat exposed to a novel It% environment becomes anxious or stressed than a rat exposed to foot-shock stimulation in that same environment; their subsequent activity when placed again in the same environment justifies such an assumption, A degree of arousal, anxiety or stress may be necessary for learning (see Refs 1, 2) but too much of either will hinder learning, both in rats and in humans (see Box). It is likely that benzodiazepines are released in different amount: in different brain structures in response to different levels of anxiety or stress. The release was greater after the more stressful avoidance procedure than after the less stressful habituation task in all structure@. The release caused by the avoidance task was quantitatively and proportionally much higher in the hippocampus and media) septum (two stru~hms related to the percep tion of, or reaction to, anxiep) than in the amygdala or cortex. However, release did not correlate with the cognitive complexity or

nature of the tasks. Thus, functions associated with the hippocampus”, such as habituation (which involves the use of working memory and possibly of a cognitive map; see Refs 2,6), and the initial execution and later inhibition of exploratory responses, were not accompanied by changes of hippocampal benzodiazepinelike immunoreactivity. These results suggest that benzodiazepine release represents a simple, general response uf the amygdala, septum and hippocampus to anxiety and/or stress, proportional to the level of anxiety and/or stress. If this were indeed the case, regulation of memory storage processes by benzodiazepine_GABAA mechanisms in different brain regions would be a consequence of the perception of, or the reaction to, anxiety or stress. It has been suggested that the modulation of post-training memory processing depends on the analysis by limbic and other structures of the affective and/or emotional content’5 of the experiences, of which the anxiogenic value- may be an important part. In some cases - for example when tasks cannot be acquired property because they are accompanied by too much anxiety’*’and are therefore open to interference by concomitant or by subsequently acquired information33 the brain benzodiazepine-GABAA systems may upreguiate rather than downregulate memory. Systemic benzodiazepines are indeed known to block retrograde interference, both in animals’? and in humans”; and a recent preliminary study on the effect of intraamygdala midazolam administration in rats has shown a depressant effect on memory by pretraining microinjection, in two different tasks, and a facilitatory effect by post-training administration in an aversive task&. These memory facilitation effects of systemic or local post-training benzodiazepine admintstratlons are both explained by d blockade of retrograde interference”*w**. A screening of the effect of intracerebral flumazenil and midazolam administration on a large number of different learning tasks should test this prediction. This study is now under way (j. H. Medina and I. Izquierdo, unpublished).

EDZ-GABA,, mechanisms and cunsolidotion It is tempting to suggest that post-training modulation by the septal, amygdaloid and hippocampal benzodiazepine-GABAA mechanisms descrfbed above is the cause of the peculiar lability of memory in the post-training period. If the neurons invofved in the consolidation process were innervated by GABAA neurons, an increase in efficiency of the latter due to the action of benzodiazepine agonists would enhance the lability of the system. In the amygdala, septum and hippocampus, neuronal systems (glutamatergic, cholinergic, etc.) involved in post-training memory processing or in activities thought to be related to memory processing (LTP, theta rhythm, etc.) are under the inhibitory control of GABAergic synapses (see Refs 1, 2,22,37,39,40). Acknowledgementa Work carried out in the authors’ laboratories was supported by grants from the Fundacao de Amparo a Pesquisa do Es&do do Rio Grande do Sul (FAPERCS) and Con&ho National de Desenvolvimento Cientifico e Teenologico (CNPq), Brazil, and Consejo National de lnvestigaciones Cimtificas y Tecnicas (CONICET), Argentina. References 1 McCaugh,). L. (19B8) in

Prrsprrtives of Mcemry Rtsturcb (Sokm~, I’. R., Coethals. C. IL, Keky, C. M. and Stephens, 8. R., eds), pp. 33-64.

!kWinner 2

l&hkb

1. (19B9) Brbev. Nrtual Eiol.

51. VI-202

3

4 5 6 7

8 9

10

I~&erdo,~ I.,

Da Cunha, C. and Medina, 1. H. (1990) Nrurosci. Biekbaa. Rev. 14.41%424 lzquierdo. I. cl al. (1990) Bchav. Neural Bid 54, 105-109 Irquierdo, I., Pet&a, M. E_and Medina, J. H. (1990) B&au. N~uraI Bid. W, 2741 Wolfman, C. ct ol. 11991) Brain Rts. 548, 74-BO Sangameswaran, L., Faks, H. M., Friedrich, P. and de Btas, A. L. (1986) I’roc. Natf Acad. Sri. USA 83.9236-9240 Wildmann, J. ct al. (1987) J. Nrural rnln,nm.70, 383-3BB De Robertis, E., Peru, C., Paladini, A. C. and Medina, 1. H. (MB) Nrwrorbtet. In!. 23, l-1 1 hkdina. J. H., Pena. C., Pivr. M., Paiadini. A. C. and De Robertis, E. \l$B~~;rm. Biopbys. Rrr. Con~nrrr.

11 Unkld,

E., Fischer, C., Rothemund, E. and Klotr, U. (1990) Biocbmr. Pkannarof. 39,210-212

TiPS - ]U~J1991[Vol. 121 12 Izquierd& 1. (1989) Treads Pl~srmacof. Sri. 10, 473476 13 Bmn, R. A. and McGaugh, J. L. (1961) J. Comp.Physkl, Psychd. 54,498-501 14 McGaugh.1. L. (1989) Brain Rn. Bull. 13, 15 Katz, R. 1. and Liebkr. L. (1978)Psychophrmrcology

!%,191-193

16 Castellano, C. and McGaugh, J. L. (1989) Behau. Nenml Biol. 51, MS-170 17 McGaugh, j. L., Casteliano, C. and Brioni, j. D. (1990) Behao. Neurosci. 104, 2b4-267 18 Brioni, j. D., Nagahara, A. H. and McCaugh, J. L. (1989) Brain Res. 47, 105-112 19 CasteUano, C., Brioni, J. D., Nagahara, A. H. and McGaugh, J. L (1989) Behav. Neal01 Biol. 52,X’&179 20 Bonanno. C., Fontana, C. and Raiteri, M. (1988) Eur. J. Phormacol. 154,22It224 21 Chrobak, J. J., Stackman, R. W. and Walsh,T. J. (1989)Behav. Neural Biol.52, 357-369 22 Brioni, J. D., Decker, M. W., Gamboa, L., fzquieedo, I. and McCaugh, J. L. (1990) Brain Res. 522.227-234 23 Thiebot, M. H. (1965) Ncumsci. Biobrhm. Reu. 9,~100 24 Lal, H., Kumar, 8. and Forster, M. J. (1968) FASEBJ. 2,2107-2711 25 Patin, M. E., fzquierdo, 1. and Madina, J. H. (1989) Broz. J. Med. Biol.

265 Rn. 22,1501-1505 26 Braestrup, C. (1988) Nearochem. Jnt. 13, 21-24 27 Jensen, L. H.. Stephens, D. N., Sarter, M. and Petersen, E. N. (1967) Brain Res. Bull. 19.359-364 28 Novas, M. L., Wolfman, C., &dins, J. H. and De Robertis, E. (1988) Phcrmacol. Biochem. Eehav. 30,331-336 29 O’Boyle, C. et al. (1983) Br. J. Anneslh. 55,349-356 30 Da Cunha, C. et al. Braz. J. Med. Biol. Res. (hi press) 31 File, S. E and Pellow, S. (19%) Psychophanacology 88, l-11 32 Slobodyansky, E., Guidotti, A., Wambebe, C., Berkovich, A. and Costa, E. (1989) J. Neurochem. 53,127~1284 33 Cahill, L., Brioni, J. D. and Izquierdo, I. (1986) Psychopharmacology 90,554-556 34 Chaws, M. L F., Peuin, S., Jardim. C. P. and Izquierdo, 1. (195Q Broz. 1. Med. Biol. Res. 23.417-421 35 Jensen, R. A., Martinez, J. L., Jr, Vasquez, B. J. and McGaugh, J. L. (1979) Psychopharmacology 64,X?&126 36 Hayes, P. E. and Kirkwood, C. K. (1989) in PhamuxoLerapy: A Rlhophysiologic Apprvach @iPiro, J. T., Albert, R. L., Hayes, P. E.. Yea. C. C. and Pusey, L. M., eda), pp. 765-781, Elsevier 37 Da Cunha, C. .eIPI. Proc. Third Int. Symp. Neum~oxins,Solis. Uruguay (in press)

Giya&iology: a growing field for drug design Karl-Anders Karlsson Recent information on protein-carbohydrateinteractionsin physiologicaland palhological situations substantiates Ike intuitive belief in carbohydrutes us candidates for drug design. Most nolcworthy, short saccharidc sequences have been shown to be specific receptors for adhesion of circulating leukocytes to vascular endolhelial cells, a phenomenoninduced al local inflammatory sites. There is strong evidence that mammalian sperm cells carry pmlcins thal interact specifically with sac&ride receptorson eggs. 771~ influenza virus rccepfor on animal cells, siulic acid, has been analysed in the cry&al conformationas a complexwith the vinf receptor-binding prohin. Thcscand other convincing examples discussed here by Karl-Anders Karlsson will inspire new approaches to the treatment of several imporfanf medical conditions where exisfing methods are insufficient. The field of carbohydrate biology at the molecular level is entering a new stage of development. Moat scientists may think of carbohydrates primarily as an energy source in food or as the structural polyaaccharides of celhdose and chitin. However, the most interK-A. Karlsson is Professor in Ilie Drpnrfmettt of Mcdfcal Biochrmislry, U&ersiIy of CiUcboq, PO Box33031. S4UO33 G&borg, Srordett.

esting carbohydrate research today is concerned with their roles as recognition sites on Cell surfaces, providing condensed information sources for various purposes (see Box). Although efforts are being made to design synthetic carbohydrate regulators for use in metabolic diseases like diabetes (e.g. the pseudotetrasaccharide acarbose developed as a glucosidase inhibitor, see Ref. 1). and although small glycosides like

38 Landfield, P. W., McGaugh, J. L. and Tus;a, R. 1. (1972) Science 175.87-89 39 CollinRridge, C. L. cI al. (19%) in The Biologj of-Memory (Squire, L.. R. and Lindenlaub, E., eds), pp. 233~~. Schattauer 40 Stewart, M. and Fox, S. E. (1990) Trends Nrurosci. 13, 163-169 41 Basile, A. S., Bulger, G. T.. Lueddens, H. W. M. and Skolnick, P. (1989) J. Pharmacol. Exp. Tker. 248.463-469 42 Gee, K. W., Brinton, R. E. and McEwen, 8. S. (i96B) J. Fltarmrcol. Exp. Thcr. 244, 379-383 43 Da Cunha, C. et al. Brrlir &es.(in press) 44 Gray. J. A. (1982) The Ncuropqrchofogy of Annrry: An En+y into thrFunctions of the Septo-Hippocampal Syslm~, Clarendon 45 Squire, L. R. (1%7) Memory and Bmin, Oxfwd University Press 46 Dickinson-Anson, H. A. and McGaugh, J. L. (1990) Proc. Fourth Conf. Ncarobiol. Learning Memory, Abstr. 6, Center for the Neurobiology of Learning and Memory, University of California, Irvine RoS4M+ I’-chlordiatPpam PKlllffi:l-(2~Maophenyl).N-methyl-N(1-mathyl-propyl)-lisoquinoline carbuxamide

cardiac glycosides and some antibiotics have been in use f&ra long time, selective carbohydratebased drugs are still rare. The purpose of this article is to provide the non-specialist with selected examples of progress within glycobiology that may lead to rational drug design. AdIKaiireceptoraon infliuMatory~ Cells associate specit%ally with each other to form solid tissues and organisms, but they also interact more dynamically in the blood and lymph circulations, requiring sensitive signdling systems and sophisticated regulation. Several families of adhesion molecules, cell surface proteins or glycoproteins that interact specifically with receptors on other cell types, have recently been identified. Of special interest for pharmacolw are recognition and adhesion phenomena of the immune system2, which are very relevant for the major clinical areas of infection, cancer, inflammation, allergy and autoimmune disease. For example, leukocytes migrate from the circuIation into tissues to kill pathogenic microorganisms during an