Neuroscience & BiobehavioralReviews, Vol. 10, pp. 449-461, 1986. AnkhoInternationalInc. Printedin the U.S.A.

0149-7634/84$3.00 + .00

Neurotransmitters, Anxiety and

Benzodiazepines: A Behavioral Review
R. A. S H E P H A R D
D e p a r t m e n t o f Psychology, University o f Ulster at Jordanstown
N e w t o w n a b b e y , County Antrim, N Ireland BT37 OQB
R e c e i v e d 12 O c t o b e r 1984
SHEPHARD, R. A. Neurotransmitters, anxiety and benzodiazepines: A behavioral review. NEUROSCI BIOBEHAV
REV 10(4) 449-461, 1986.--The possible involvement of serotonin, GABA and opioid peptides in anxiety and in the
mechanism of action of benzodiazepine tranquillizers have recently been the subjects of intensive biochemical, neurophysiological and behavioral research. The present review examines the behavioral evidence, viewing anxiety and benzodiazepine action as far as possible separately. Four behavioral paradigms of experimental anxiety or "conflict behaviors" are described and assessed for soundness with some practical considerations. The functional significance and
pharmacology of benzodiazepine receptors are discussed, and the cases for a number of putative endogenous ligands are
examined. Conflict behavior is attenuated by drugs which reduce functional serotonin activity and enhanced by serotonin
agonists, but there is little evidence to implicate serotonin in benzodiazepine action. GABA antagonists both intensify
conflict and reduce benzodiazepine effects, but evidence of the reverse effects with GABA agonists is more equivocal. The
interpretation of behavioral effects of opiate agonists and antagonists and their interactions with benzodiazepines is
hindered by their actions on motivational systems other than anxiety, and evidence for an important role of opioid peptides
is only suggestive. Some promising lines for future research are indicated.
Anxiety

Benzodiazepines

Serotonin

GABA

Opiates

B E N Z O D I A Z E P I N E (BZ) tranquillizers were introduced

into medicine some 25 years ago, rapidly established themselves as useful in the therapy of anxiety states and took a
place amongst the most widely prescribed drugs in medicine
[89,143]. Initially, progress in analysing the mechanism of
BZ action in terms of neurotransmission and other brain
processes lagged behind their therapeutic success and little
of current significance remains from the 1960s. By 1975,
however, a number of tenable hypotheses of BZ action were
extant [33, 34, 50, 91, 165, 175], and a further stimulus to
research in this area followed shortly when specific receptors were described [171]. Subsequently, research on the
mechanism of BZ action has proceeded rapidly on biochemical, neurophysiologicai and behavioral fronts. However, for
the former two lines of enquiry, it could be argued that if the
emerging hypotheses of BZ action are eventually to result in
major advances in the therapy of anxiety, then it must be
possible to derive predictions which are testable initially in
animal behavioral models of anxiety, and ultimately in humans. For example, if the effects of BZs on anxiety could be
attributed to interference with a particular neurotransmitter
system, say, by reducing the release of the substance from
neurons, then a different kind of intervention in this
hypothetical system, say, blocking the post-synaptic receptors, should produce a more effective anxiolysis. One purpose of this review is to assess the extent to which behavioral data support current views of the mechanism of BZ
action on anxiety, other BZ actions being considered only
where they are essential to understanding the background or
interpretation of such results.

Conflict behavior

A further point which should be made is that analysing the

neurochemistry of BZ action and of anxiety per se, are logically separate enquiries, though it is not unreasonable to
expect some connection. These matters are often confounded in the literature and the distinction is much more
than a semantic one but has both practical and methodological implications. Practically, it is quite possible to conceive
of a situation in which BZ action is fully understood yet is
found to be only a part of the biology of anxiety, indeed this
might almost be expected from the inability of BZs to completely resolve all clinical anxiety states. The author naturally hopes that BZ research will lead to the development of
anxiolytics of greater effectiveness than those available at
present, and it is most unlikely that such drugs would be
chemically BZs or even that they would act only at the BZ
receptor because full agonists at a given receptor have the
same maximal effect. Consequently, research on the neurochemistry and neurophysiology of anxiety should continue
to have a broad base and not be exclusively concerned with
mechanisms of BZ action. Methodologically, studies of the
capacity of such drugs as serotonin antagonists, GABA
agonists or opioids to attenuate experimental anxiety must
be regarded as bearing on the neurochemistry of anxiety.
Even if the behavioral effect does resemble that of BZs,
comparisons of this sort do not of themselves reveal anything about the mechanism of BZ action since the neurotransmitter intervention might affect anxiety processes in a
way entirely independent of BZ systems. Behavioral studies
of the mechanism of BZ action, on the other hand, generally
assess interactions of BZs, or latterly various BZ antagonists

449

450

SHEPHARD

[66], with drugs affecting particular neurotransmitter systems in known ways such as agonists or antagonists at postsynaptic receptors. This article, then, will attempt to review
the involvement of certain neurotransmitters, especially
serotonin, G A B A and opioid peptides, in anxiety and BZ
action as separate questions, though many studies quite justifiably bear upon both.
BEHAVIORAL PARADIGMS OF ANXIETY

Although the behavioral pharmacology of BZs has been

extensively reviewed [34, 37, 53, 83, 92, 99, 173-175, 190,
191], the intention of this article is to examine chiefly anxiety, and therefore most of the evidence cited here will have
employed established animal models of anxiety and of
anxiolytic drug action. As will be seen, such models generally involve bringing two motivational systems, each of
which would alone direct opposite behavior, into conflict,
and are therefore often termed conflict behaviors. Although
there are many intuitive, phylogenetic and pharmacological
reasons (e.g., [87]) for regarding these animal behaviors as
good models of human anxiety, for the purposes of this review reference will be made primarily to the physiology and
pharmacology of conflict behavior, with " a n x i e t y " used
only as an explanatory concept or to describe human emotion. The criteria for, and range of, conflict behaviors should
therefore be pointed out at this stage. Five criteria would
seem fairly uncontroversial, these being:
Specificity. Effects on the behavior in question should be
produced only by BZs and those agents which have some
similar pharmacological actions, e.g., barbiturates, meprobamate, alcohol, serotonin antagonists and G A B A
agonists. It follows from this that suitable paradigms are
likely to show facilitatory effects of BZs on behavior since
behavioral inhibition can be produced by virtually any drug
at appropriate doses, as well as by a range of nonpharmacological treatments.
Sensitivity. One would expect suitable anxiety models to be
sensitive to low doses of BZs. Although it may not be appropriate to be too dogmatic about this, especially in view of
wide differences in potency of certain BZs according to vehicle [52] and route [147] of administration, one would certainly expect to detect the effects of 5 mg/kg chlordiazepoxide, for example.
Correlation with clinical potency. Although, as is usual in
psychopharmacology, doses per unit body weight are generally substantially higher in studies of conflict behavior in
animals than in clinical practice, it should be expected that
strong positive correlations be observed between relative
clinical activity of a series of anxiolytics and their effects on
the animal model.
Absence of, or reverse, tolerance. Conventional wisdom
based on both clinical [187] and animal [116] studies holds
that while certain actions of BZs, such as sedative ones,
show tolerance, anxiolytic action does not, and may even
increase with repeated doses. Whilst the continued clinical
efficacy of BZs following months or even years of continuous treatment is being questioned, this does not justify regarding behavioral paradigms in which tolerance is observed
after a few daily injections as suitable pharmacological assays of conflict behavior.
Validity. This term refers to the resemblance between the
methodology of testing and the construct which is supposed
to be measured, in this case anxiety. Psychopharmacologists
interested in anxiety are perhaps especially fortunate in hay-

ing a number of procedures available for which this resemblance seems good. However, since BZs have been reported to have direct actions on some motivational systems
used in assessing conflict behavior, such as feeding [37.40.
41] and nociception [53], the possibility that the behavioral
effects of BZ might be due to such actions rather than on the
inferred conflict or " a n x i e t y " deserves careful consideration.
Fortunately, at least four well established paradigms
seem to satisfy the first four criteria and these will be briefly
reviewed, focussing on the extent to which they fulfil the
fifth and on practical considerations.
Punished operant behavior. A multiple schedule was described in 1960 in which periods of partial reinforcement of
operant behavior (providing a test for non-specific drug effects, e.g., sedation) were alternated with periods in which
every response was rewarded with food, but also accompanied by electric shock [80]. By appropriate adjustment of
shock severity, it is possible to obtain varying degrees of
behavioral suppression during these periods. In the initial
report barbiturates and meprobamate [80], and shortly afterwards BZs [76,79] were shown to enhance the rate of such
punished responding. This effect was attributed to anxiolytic
actions of these drugs, which were suggested to resolve the
conflict inherent in the paradigm. A minor variant in which
every tenth response of the subject is simultaneously rewarded and punished has been suggested to be less sensitive
to variations in levels of shock and food deprivation [56], and
despite the scarcity of hard data supporting this claim, has
also entered general use. Punished operant behavior remains
the standard animal conflict paradigm against which others
should be compared.
BZ-induced increases in punished behavior could,
theoretically, be due to reduction of the effectiveness of aversive stimuli. However, this does not appear to explain this
behavioral action since opiates do not fully simulate this BZ
effect [77,117], and even disconnection of the stock
generator produces only slow recovery of responding
whereas BZ effects are rapid in onset [53]. The possibility
that BZ effects on appetite may explain increases in punished responding is considered in a later section.
With sufficient training, animals will arrive at a control
baseline of responding which is almost invariable between
days. Consequently, if, following a drug administration, an
animal shows a dramatically increased rate of punished responding, then this would be good evidence that the drug can
exert anti-conflict effects. Unfortunately, the questions
which psychopharmacologists ask are rarely so simple, If
one wishes, for example, to study drug interactions, compare different agents or examine differences in drug responsivity between strains of animals, then one requires a quantitative measure. Not only do BZs increase mean punished
response rates but they also increase variability. Consequently, substantial numbers of subjects and of observations
per subject, as well as scalar transformations [159], may be
required for statistically-reliable results. Taken together with
the training time required, experimenters who do not have
extremely well-equipped laboratories might, if for no other
reason, consider using other conflict paradigms on parsimonious grounds.
Punished drinking behavior. Use of equipment which permits the licking of rats at a water tube to be monitored enabled a water lick conflict test for BZ action to be developed
[186]. This paradigm is analogous to that reviewed above in
that deprived animals are punished by electric shock, in this

TRANSMITTERS, ANXIETY AND BENZODIAZEPINES

451

case for consummatory rather than operant responses and

that BZs increase such responding. Initially this procedure
was described for experimentally-naive subjects [ 186], which
eliminates training time but requires large numbers of
animals to be used in studies. However, more recently [133],
drug effects have been shown to be even more reliable in
test-experienced subjects, which implies that useful results
can be obtained from fewer subjects, without the extensive
training required to examine punished operant behavior. One
does, of course, lose some of the experimental control of
subjects' behavior, and the possibility of adding extra components to the schedule. F o r the reasons given in the above
section there seems little reason to suspect BZ actions on
nociception of producing their behavioral effects. However,
in view of the growing evidence of direct enhancement of
drinking with BZs [40,41] some caution in invoking the construct of anxiety to explain BZ enhancement of punished
drinking seems indicated.
Neophobia. This term refers to the inhibition of (usually)
feeding due to novelty and is further divisible into inhibition
due to novel food (food neophobia) and that due to the unfamiliar test situation and procedure (apparatus neophobia).
Following its initial description as a conflict behavior [137], it
soon became apparent that both types of inhibition can be
attenuated by BZs [ 176]. The conflict in this paradigm can be
regarded as being between the hungry animals desire for
food and its fear of novelty.
For some time, the extent to which this test measures
anticonflict drug actions as against their effects on appetite
has been controversial, though quite why analogous questions have not been raised to the same extent in relation to
punished operant behavior is unclear to this author, especially since enhanced food deprivation has been reported to
increase punished responding [3, 4, 54, 118, 119], as do BZs.
However, the doubts have generally been raised in the context of this paradigm and will be surveyed here. The evidence for an appetitive interpretation of BZ enhancement of
eating in novel situations essentially rests upon three planks,
and close examination of each of these reveals flaws.
The most fundamental of these is, of course, the proposition that BZs can exert appetitive actions, that is to say can
enhance food intake in the absence of any suppressive influence. Since rats have been used almost exclusively in
neophobia studies we will, for the time being, confine discussion to this species. Since inhibition of feeding in rats can be
elicited very readily by factors as subtle as, for example, novel
food containers [184] and even changed position of the food [8],
detailed examination of the methods employed is necessary
to conclude that appetitive effects have been demonstrated.
Such examination may prove impossible, reveal factors
which are well known to inhibit food intake in rats, such as
isolation of the subjects [5,44], stressful injection procedures
] 172] or absence of a direct measure of amount of food eaten
[38], and few reports survive close analysis [149,192]. Set
against these are negative reports [25, 43, 97, 102], plus the
possibility that many such nonsignificant negative findings
have gone unpublished. The author has been unable to
enhance home-cage feeding of rats with BZs [153] and would
be interested to hear of unpublished data bearing on this
question in either direction. Although evidence from other
species [37,73] indicates that BZs may elicit appetitive actions in some circumstances, the data for the major species
used in conflict studies are less than conclusive.
The second plank is formed of studies which demonstrate
similarities between effects of BZs on feeding and those of

food deprivation. Diazepam has been reported to induce

feeding in non-deprived rats [192], and more detailed resemblances between the feeding behaviors elicited by BZs
and by food deprivation have been reported subsequently
[29, 37, 44]. However, it is well known that increased deprivation only enhances motivation up to a point and, in the
case of rat feeding, this point seems to be reached at about 24
hours [19, 30, 156], especially if the animals are meal-fed. It
is therefore difficult to see how BZs and related drugs could
consistently produce increased feeding in novel situations in
strongly deprived and meal-fed rats [153-157, 160, 161, 167]
by an appetitive mechanism. Moreover, further increasing
food deprivation, by omitting the meal on the pre-test day in
meal-fed rats, has no effect on feeding in a novel situation or
BZ action [156]. If BZ effects on food-motivated conflict
behaviors depended on appetitive mechanisms, one would
expect their action to be strongest in satiated subjects, and
weaker as the maximally-effective level of food deprivation
is approached. F o r neophobia, chlordiazepoxide produced
greater enhancement of feeding in meal-fed rats tested some
24 hours after their last meal than in satiated ones tested
some 2 hours after their large daily meal [160]. Similarly, for
punished operant behavior, chlordiazepoxide enhanced punished responding when subjects were at 70% or 9(F/b, but not
100% of their flee-feeding weight [119]. These studies do not
support appetitive interpretation of BZ action but rather
suggest that the magnitude of drug action is proportional to
the degree of conflict. Lastly, other motivational manipulations of feeding, such as lateral hypothalamic stimulation
[166] and lesions in the ventromedial hypothalamus [129,167]
do not apparently either simulate or attenuate BZ action.
Thirdly, when rats are presented with a choice of novel
and familiar foods relatively high doses of BZs enhance
choice of the former, whereas lower doses (and food deprivation) increase preference for familiar foods [29, 37, 38, 42,
45]. Despite small variations in the doses of BZs which elicit
these effects, this observation seems robust [97,161]. What is
contested, however, is the assertion that drug enhancement
of familiar food intake reflects appetitive actions of BZs and
that only increased eating of the novel food can be regarded
as anti-conflict drug action [37, 38, 45]. Since the rats used
were naive with respect to several aspects of the test environment and procedure, which have long been known to
inhibit feeding [93], it hardly seems implausible that BZ
enhancement of familiar food intake is also due to
anticonflict effects. This hypothesis has been tested by comparing naive groups of rats with subjects which have been
carefully acclimatized to all aspects of the experimental
apparatus and procedure. Not only did acclimatization
produce a massive enhancement of feeding, but it also prevented BZ effects [160], suggesting a major involvement of
apparatus neophobia in determining behavior and BZ effects
in this paradigm.
Despite the defense above, it is not the author's intention
to represent neophobia as the answer to every experimenter's needs in this area. Some subjects fail to eat in the
necessarily pre-determined test period, which creates statistical problems especially since the proportion of non-eaters
may increase directly with BZ dose [167]. Meal-feeding,
however, largely overcomes this problem [153], but at a cost
in time. From an anthropomorphic point of view, moreover,
a schedule of food deprivation followed by injection and
placement in a novel environment with unfamiliar food seem
rather salient events in the uninteresting lifetime of the laboratory rat. A novel situation is really only so once and it

452
cannot be recommended that a rat participates in more than
one such study for this reason alone, quite apart from possible persistence of drug action, enzyme induction and so on.
Therefore, some of the advantages which neophobia offers in
terms of limited pre-experimental work and cheap and infallible apparatus have to be set against the requirement for
large numbers of naive subjects per experiment.
Social interaction. This paradigm consists of assessing the
interactions between the experimental animal and an undrugged companion in a novel test environment, such social
interactions being increased by BZs [68,69]. Like neophobia,
it utilises rats' aversion to novelty as an inhibitory behavioral
influence, with the attendant problems concerning repeated
observations. It has also been pointed out that tests involving predictable punishment of behavior 'fail to encompass a
potent cause of anxiety, uncertainty' ([68], p. 23) and that,
while clinical anxiety must be regarded as maladaptive, there
seems nothing maladaptive about the passive avoidance of
electric shock [153]. Social interaction and neophobia may,
therefore, show a resemblance to clinical anxiety which is
theoretically superior to that of punished behavior. However, predictability is not a necessary feature of punishment
procedures, this does not seem the place to revive the old
controversy about the natures of ~anxiety' and ~fear' and
major differences in the pharmacology of these types of test
have not become apparent. The fact that social interaction
does not measure behavior directed towards food or water
may also be construed as an advantage over the preceding
tests depending, in part, upon one's interpretation of the
above discussion.
BZ RECEPTORS, EXOGENOUS AND PUTATIVE ENDOGENOUS
LIGANDS
The existence of brain-specific BZ binding sites is by now
well known and they meet criteria necessary to designate
them receptors. As will be seen, non-benzodiazepine compounds undoubtedly interact with these, but this review will
follow the conventional nomenclature and refer to them as
BZ receptors. Since this review focusses on relationships
between biochemistry and behavior, the reader is referred
elsewhere (e.g., [142,170]) for accounts of the discovery and
structure of BZ receptors. There are, however, a few general
observations of behavioral significance best made here. In
the first place, it quickly became clear that the distribution of
BZ receptors across species [128] and brain regions [21, 23,
163, 195] bore no close relationship to those of any established neurotransmitter. BZ receptors must therefore be
either wholly independent of established neurotransmitter
systems or, as now seems more likely, have a selective physiological role at certain synapses employing a particular neurotransmitter or neurotransmitters. Secondly, reports of an
association between number of BZ receptors and emotionality in different strains of rats [82,145] and mice [144], were
not supported by large-scale investigations of genotypes derived from the Roman strains of rats [162,163], despite the
fact these show large variance in BZ receptors and behavior,
and even the association between BZ receptor binding and
behavioral sensitivity to diazepam has been questioned
[162]. There are also large sex differences in BZ receptors in
several brain regions [163] which show no obvious correlation with sex differences in behavior [85] or neurophysiology
[61]. It is therefore clear that anxiety is determined, at least
partly, by factors other than BZ receptors, implicating other
neurotransmitter systems.
Exogenous ligands or drugs acting at the BZ receptor are

SHEPHARD
now usually divided into "agonists" (substances which arc. or
resemble pharmacologically, clinically-used BZ anxiolytics).
~reverse agonists' (substances which elicit effects essentially
opposite to those of the agonists) and 'antagonists' (substances which prevent the actions of one or both of the above
but have relatively little intrinsic activity). This taxonomy is
open to criticism because the term agonist is generally used
to refer to agents which mimick the actions of established
neurotransmitters; since no such substance has been identified for the BZ system it could be an 'anxiolysin." an
"anxiogenin' (implying that, say, diazepam is an antagonist),
or not exist at all. Moreover, in other neurotransmitter/receptor systems the term antagonist seems to cover both
'antagonists' and 'reverse agonists' as used in the BZ context. Nevertheless, no satisfactory alternative nomenclature
has yet been developed.
The agonists comprise at present the familiar anxiolytics
such as chlordiazepoxide and diazepam, and a number of
non-BZ substances [1,32, I 11, 194]. Amongst the latter, one
compound, CL218872, is perhaps of particular interest because it discriminates between subtypes of brain BZ receptor, which seem to have differential neuroanatomical distributions [196] and ontogenetic development [112]. The major
behavioral, and potentially clinical, implication of such
selectivity is that it may prove possible to dissociate the
anxiolytic properties of drugs acting on the BZ receptor fi-om
sedative ones. Such a claim has been made for CL218872 [ 1].
but has also been disputed [130].
The reverse agonists include some BZ structures, but
most interest has focussed on the /%carbolines of which a
number have been reported to provoke anxiety in humans
[22], or to intensify behavioral suppression in animal conflict
procedures. Thus, /~-carbolines reduce social interaction
[71], enhance the effectiveness of low levels of shock in suppressing drinking [47], and reduce rates of punished operant
responding [60]. These effects can be attenuated by BZ
agonists or antagonists, which together with biochemical
data, strongly suggest that they are mediated through the BZ
receptor. The physiological significance of these findings is
discussed below.
The benzodiazepine antagonists are exemplified by
RO15-1788 which binds to all neural BZ receptors [142]. Almost all behavioral studies on this class of drugs have employed this compound. In humans it reduces anxiolytic effects of BZs [55] and has been useful in comatose patients
following BZ overdose [152]. RO15-1788 also antagonises
the actions of BZs on punished behavior in rats [20]. Perhaps
most interestingly, RO15-1788 has also been reported to
antagonise the actions of the reverse agonist /3-carbolines
[47,71], Therefore it may be a kind of neutral ligand at benzodiazepine receptors occupying the binding site and preventing either agonist or reverse agonist actions. Although
RO15-1788 may have some partial agonist properties
[66,185], others have been unable to detect these [20, 147,
164, 181] or have only done so at doses much higher than
those necessary to antagonise BZ effects. Whether or not
RO15-1788 has such properties may not be of great theoretical significance since drugs acting at the BZ receptor can
probably be regarded as a continuum from full agonists
through partial agonists and antagonists to reverse agonists.
The precedent of the opiate receptor/peptide system directed speculation on possible endogenous ligands for BZ
receptors almost as soon as the latter were discovered. Unfortunately, the search for this substance has not eventuated
on its rapid elucidation and contradictory and hasty claims

TRANSMITTERS, A N X I E T Y AND B E N Z O D I A Z E P I N E S
have been made. Three classes of compound have received
serious attention as candidates for this role, the/3-carbolines,
proteins/peptides and purines.
Urine contains many neurotransmitter metabolites. When
the fl-carbolines were obtained by acid hydrolysis of human
or animal urine and shown to interact potently with the BZ
receptor, speculation was naturally on the possibility of a
neurotransmitter role for fl-carbolines at the BZ receptor.
This hypothesis has been strongly criticised [170], largely on
the grounds that acid hydrolysis of a wide variety of
trytophan-containing proteins produces fl-carbolines and
that their precursors in urine are heterogeneous, which does
not suggest that these urinary substances are metabolites of
fl-carbolines. Moreover, since no biosynthetic mechanism
for their production in the brain has been postulated, there is
no reason to regard them as directly involved in the physiology of anxiety, though they are of considerable pharmacological interest, as noted earlier.
A large number of proteins or peptides have also been
postulated as endogenous ligands at BZ receptors (see [170]
for review). For the most part, these have been large
molecules and therefore difficult to characterise. However,
some relatively small peptides have been isolated, and have
anti-conflict activity [58]. Squires [170] has hypothesised
that many peptides or proteins reported to be active at the
BZ receptor may be fragments of the receptor itself, and
therefore artifacts of the extraction techniques. At present
this argument cannot be refuted for any of the candidate
endogenous ligands in this category.
A number of purines, including caffeine, have been reported to inhibit diazepam binding at BZ receptors [2,115].
Although the affinity of these is probably too low for them to
be regarded as likely neurotransmitter substances at BZ receptors, these might explain the rather paradoxical effects of
caffeine, which has been reported both to have anti-conflict
effects and to antagonise diazepam action [18,28]. Thus caffeine might be a non-potent partial agonist at BZ receptors.
By far the most promising purine as a putative endogenous
ligand would seem to be 1-methylisoguanosine which is
naturally-occurring, has a relatively high affinity for BZ receptors and has muscle-relaxant properties [57]. It has
proved difficult to assess the effects of this compound on
conflict behavior, because of its powerful muscle-relaxant
properties I57]. Examination of the interaction of 1-methylisoguanosine with BZ receptor, and other, antagonists would
be of considerable interest.
In conclusion, there are intuitive reasons for suspecting
that an endogenous neurotransmitter has a physiological role
at BZ receptors. Moreover, the existence of both agonists
and reverse agonists in this system suggests that there must
be some physiological activity at the BZ receptor complex,
though this need not necessarily be at the BZ binding site.
However, no such neurotransmitter has yet been identified
and, even if it were, this would not preclude an involvement
of other neurotransmitters in BZ action, let alone anxiety.
I N V O L V E M E N T OF SEROTONIN 1N ANXIETY AND BZ ACTION

By 1975, a considerable volume of both behavioral and

biochemical data suggested involvement of serotonin in the
physiology of anxiety and, moreover, that the behavioral
effects of BZs might be due to actions on this neurotransmitter system [175, 190, 191]. Indeed more behavioral evidence
related serotonin to conflict behavior and BZ action than any
other neurotransmitter at that time. Since then, however,

453
interest in these questions seems to have reduced. This decline may be attributable to the lack of any direct involvement
of serotonin in the BZ receptor system. Nonetheless,
serotonin is not reviewed here merely for completeness, but
because the involvement of neurotransmitters in anxiety and
in the mechanism of BZ action are here being viewed as far
as possible separately. As will be seen, there is little direct
evidence for the latter but the case for involvement of
serotonin in conflict behavior is strong, and this will be surveyed first.
Anti-conflict effects of antagonists at post-synaptic
serotonin receptors were first suggested by the capacity of
methysergide and 2-bromo lysergic acid diethylamide to increase punished responding in pigeons [84]. In a separate
session, these drugs produced only a small enhancement of
unpunished behavior. Similar results were subsequently reported for methysergide and cinanserin using the conventional multiple schedule and rats as subjects [175].
Methysergide also attenuates neophobia [157,158] as do certain fl-adrenoceptor antagonists [157,158] by a mechanism
which probably depends upon antagonism at central serotonin receptors [88, 121,150, 158, 189]. The serotonin selective
neurotoxins 5,6 and 5,7-dihydroxytryptamine have also both
been reported to enhance punished operant behavior
[175,183], although there is also a negative report for the 5,7
compound [181]. The tryptophan hydroxylase inhibitor
parachlorophenylalanine (pCPA), also has anti-conflict effects [78, 96, 146, 159, 191] which have not invariably been
confirmed [15,35]. The inconsistencies have been suggested
to be due to use of statistical analysis [15] or less frequently
punished behavior [35] in the negative reports, but both have
been used in studies showing increased punished behavior
with pCPA [96,159]. Although pCPA is known to affect other
neurochemical systems, the reversal of its anti-conflict effects by the serotonin precursor 5-hydroxytryptophan
[78,96] and by the direct serotonin receptor agonist
5-methoxy-N,N-dimethyitryptamine or 5-MeODMT [159]
suggests the serotonergic system as the probable locus of
action. Therefore, drugs reducing the functional activity of
serotonergic systems, whether by receptor antagonism,
neurotoxicity or synthesis prevention seem to elicit anticonflict effects.
Conversely, a-methytryptamine, a long-lasting agonist at
serotonin receptors, intensifies behavioral suppression induced by punishment in pigeons [84] and rats [175]. Furthermore, intraventricular injections of serotonin itself inhibit further punished behavior and antagonise the anticonflict effects of oxazepam [175]. Chemical stimulation of
the serotonergic dorsal raph6 cells with carbachol elicits
similar effects [175]. Unfortunately, most of these reports
also show increases in functional serotonergic activity to inhibit unpunished behavior as well and therefore it is difficult
to regard these effects as being specific to conflict behavior.
As has already been pointed out, drawing specific conclusions from inhibitory drug effects requires careful justification. Further caution might be taken from the pharmacological profile of 5-MeODMT, which has been shown to inhibit
feeding in novel situations [154, 156-158] and unpunished
operant behavior [159] but fails to inhibit punished responding [159]. Moreover, certain drugs generally regarded as
agonists at post-synaptic serotonin receptors paradoxically
produce anti-conflict effects [151]. However, in humans hallucinogenic experiences are well known to be anxietyproducing in variable degree and the mechanism of action of
hallucinogenic drugs is now generally thought to be due to

454

SHEPHARD

their agonist properties at serotonin, or some closely related,

receptor system (e.g., [9,107]. In view of this and the evidence above, it seems reasonable to conclude that serotonin
is an anxiogenic neurotransmitter.
The suggestion that effects on the serotonin system constitute the mechanism of action of BZs, however, can no
longer really be regarded as tenable. Originally [ 175, 190, 191[,
it was based partly upon the resemblances between behavioral effects of BZs and those of drugs which reduce functional serotonin activity and the opposite effects of serotonin
agonists. Although shown above to be fairly secure, this
does not directly link serotonin with BZ action, since the
serotonin and BZ receptor systems might have independent
effects on anxiety mechanisms. The other major line of evidence was biochemical [48] and neurophysiological [139]
studies which suggested that BZs reduced serotonin turnover, and that such action did not show tolerance [48,175].
Other studies, however, have failed to detect such changes
[33], or have suggested alternative interpretations [31,101].
Even ifBZs do reduce serotonin turnover, it is not at all clear
how such action would explain their behavioral effects. F o r
example, certain BZs produce serotonin agonist-like head
twitches in mice [126] and all tested potentiated those
produced by mescaline or intracerebral serotonin [127]. If
reduction in serotonin turnover were responsible for the
anti-conflict effects of BZs, then one could predict interactions between BZs and serotonergic agents. Thus, BZs
should not be effective in the presence of directly-acting
stimulants of post-synaptic serotonin receptors since these
reduce serotonin turnover themselves, and furthermore, excitation of post-synaptic receptors should render changes in
turnover unimportant since few receptors would be available
for occupation by the neurotransmitter. Similarly, anticonflict effects of BZs should be prevented by gross functional impairment of the serotonin system induced by such
things as pCPA or 5,7-dihydroxytryptamine although, since
the latter induce anti-conflict effects themselves, there is risk
of this prediction being confirmed artifactually by a 'ceiling'
effect. However, studies of neophobia [154], punished drinking [106] and punished operant behavior [159,181] do not
confirm either of these predictions. Even at doses sufficient to
induce stereotyped behavior and to inhibit unpunished operant responding, the post-synaptic serotonin receptor agonist
5-MeODMT does not affect the anti-conflict effects of
chlordiazepoxide [159]. Generalized depletion of serotonin
with systemic pCPA or localised lesions at appropriate sites
with 5,7-dihydroxytryptamine fail to prevent significant
anti-conflict effects of chlordiazepoxide [159] and diazepam
[181] respectively. Although others have reported inhibition
of BZ action with pCPA [34] and 5,7-dihydroxytryptamine
[183], excessive sedation, inadequate statistical analysis and
ceiling effects probably explain the apparent discrepencies
[159,181]. Furthermore, microinjections of RO15-1788 into
the dorsal raph~ block the reductions in serotonin turnover
produced by diazepam, without antagonising its anti-conflict
actions. Since a wide variety of treatments which would
either prevent, or render unimportant, effects of BZs on
serotonin turnover do not seem to modify their anti-conflict
actions, any capacity of BZs to reduce serotonin turnover
seems irrelevant to their anxiolytic properties.
INVOLVEMENT OF GABA IN ANXIETY AND BZ ACTION
The capacity of BZs to produce sedative and anticonvulsant effects, together with a variety of behavioral, neuro-

physiological and biochemical studies reviewed elsewhere

[10, 41, 49-51, 91, 92], implicate G A B A in anxiety and the
mechanism of BZ action. Perhaps most salient of these is the
direct evidence of G A B A involvement in BZ receptor binding, which is facilitated by G A B A agonists, but inhibited by
its antagonists [24,178]. Such observations suggest that
G A B A agonists should simulate behavioral effects of BZs
and potentiate their action, whilst G A B A antagonists should
induce essentially opposite effects. The extent to which behavioral evidence has lagged behind the predictions which
follow from biochemical and neurophysiological studies in
this area has, at times, bordered on the spectacular. However, the gap has narrowed somewhat lately, as we will see,
looking first at the involvement of GABA in anxiety.
The most impressive behavioral evidence for an involvement of GABA in conflict behavior would be if drugs enhancing the functional activity of G A B A systems could be shown
to produce anticonflict effects. Drugs which inhibit G A B A
transaminase, and thereby elevate brain levels of GABA,
including amino-oxyacetic acid [141,183], y-vinyl G A B A
[141] and ethanolamine-O-sulfate [95], fail to produce such
effects. Moreover, directly acting G A B A agonists such
as muscimol [149,179], progabide [149] and 4, 5, 6,
7-tetrahydroisoxazolo (5, 4-C) pyridin-3-ol or THIP [141]
similarly do not simulate various BZ actions. However these
negative reports may, at least in part, be due to poor penetration of the compounds to the brain since intra-cerebral
administration of muscimol [49] or G A B A itself [140,1801
produces some anticonflict actions. Ethanolamine-O-sulfate
has also been reported to be as efficacious as chlordiazepoxide in conflict tests, and more so than functional
serotonergic inhibition [96].
Despite these inconsistencies, the putative GABA agonist
valproate reproducibly elicits anti-conflict effects in several
paradigms following systemic administration [109, 110, 133,
141, 161, 164]. In the case of food-motivated behaviors, it
seems reasonable to discount explanations in terms of appetite stimulation since both indirectly [36, 98, 131] and
directly-acting [16,361 G A B A agonists produce anorectic effects following systematic administration. Unfortunately,
the neurochemical mechanism which mediate this effect of valproate are by no means clear. Valproate can inhibit G A B A
transaminase and thereby elevate total brain G A B A concentration at high dose levels [135], but elicits both anticonvulsant [135] and anti-conflict [141] actions at doses which do
not alter brain G A B A levels. A direct agonist action at
G A B A receptors is suggested by antagonism of its anticonflict effects by bicuculline [141] and picrotoxin [164] the
latter being clearly competitive antagonism suggesting a
common site of action. However, unlike other G A B A
agonists, it does not stimulate BZ receptor binding and indeed inhibits muscimol-enhanced BZ binding at high concentrations [182], though this may be due to attenuation of muscimol stimulation, rather than BZ binding itself and therefore
refect effects of valproate at GABA or BZ receptors. Comparative neurophysiological studies of G A B A and valproate
suggest some direct G A B A agonist actions of vatproate [6],
potentiation of G A B A action with rather lower amounts
[6,81], but largely antagonistic effects of valproate at G A B A
receptors in the hypothalamus [7]. Since G A B A may function as an inhibitor of feeding in the lateral hypothalamus
[103-105, 123], it would be tempting to speculate that this
paradoxical G A B A antagonist effect of valproate in a "feeding centre' of the hypothalamus might explain its effects on
neophobia [161,164] except that it is antagonised by pic-

TRANSMITTERS, ANXIETY AND BENZODIAZEPINES

455

rotoxin [164] and that anti-conflict actions of valproate are

also shown in other tests [109, I10, 133, 141]. It therefore
seems that certain putative G A B A agonists, especially valproate, can produce attenuation of conflict behavior. However, the mechanisms involved in such reductions are not
entirely clear and further work, including studies of G A B A
agonists following local administrations, seems strongly
indicated.
Several studies have shown that G A B A antagonist, picrotoxin, to induce effects essentially opposite to the behavioral actions of BZs, that is, to reduce further behavior suppressed by punishment or novelty [70, 155, 164, 169, 175].
However, caution in inferring a role for G A B A in conflict
behavior from such evidence is clearly indicated since, as
was the case for studies with serotonin agonists reviewed
above, there is little guarantee of the specificity of such effects.
Turning to the interactions of BZs with GABAergic
drugs, there is very little behavioral confirmation of the
synergism between G A B A agonists and BZs which would be
predicted from both biochemical [24,178] and neurophysiological [75,91] studies. An investigation of this point not
only failed to demonstrate enhancement of the anti-conflict
actions of a marginally-effective dose of diazepam (0.5 mg/kg)
with muscimol, but actually suggested some antagonism
[179]. BZs have also been found to block muscimol-induced
myoclonic jerks in the mouse [120]. Although the putative
G A B A agonist valproate exerts anti-conflict actions alone,
as reviewed above, it also inhibits the effects of chlordiazepoxide on neophobia [161], though in view of the ambiguities surrounding the mechanism of action of valproate,
this might be interpreted as an idiosyncratic interaction
possibly involving partial agonist properties of valproate, at
the GABA/BZ receptor complex [161] rather than reflecting
generally on relationships between these systems. Whilst
these studies do support some such relationships they do not
conform well with the above prediction of mutual synergism
which was derived from biochemical and neurophysiological
data. However, some behavioral studies can be regarded as
so conforming more closely. The G A B A transaminase inhibitor ethanolamine-O-sulfate potentiates some effects of
chlordiazepoxide on conflict behaviors [95,96], which
suggests a synergistic interaction between G A B A and BZs.
Moreover, the antineophobic effects of valproate are prevented by the benzodiazepine antagonist RO15-1788, apparently a non-competitive antagonism and therefore consistent
with there being G A B A and BZ sites on a receptor complex
since it is not overcome by increasing the valproate dose
[164]. Turning to interactions between G A B A antagonists
and BZs, a number of studies have shown some antagonism
between BZs and picrotoxin [11, 155, 175]. Since picrotoxin
generally shows intrinsic actions opposite to BZs, and as the
antagonism observed is not always consistent across behavioral measures [155], it is nonetheless difficult to assess the
pharmacological interactions involved. However, bicuculline has been reported to block antineophobic effects of
diazepam in hamsters at doses too low to affect this behavior
alone [13], suggesting some specificity for this antagonism.
Taking these lines of evidence together, there is considerable
behavioral evidence for an involvement of G A B A in both
conflict behavior and BZ action. However, there remains an
appreciable gap between biochemical/neurophysiological
studies and behavioral ones and also several apparently
anomalous findings and further research on all three fronts is
required,

I N V O L V E M E N T OF OPIOID PEPTIDES IN A N X I E T Y AND BZ ACTION

The relationships between opioid peptides (or exogenous

agents acting as agonists or antagonists at opiate receptors),
anxiety and BZ action have recently become the subjects of
intensive investigation. As far as studies of conflict behavior
are concerned, interpretation is generally somewhat confused by the fact that opiates (and to only a slightly lesser
extent their antagonists) have direct effects on virtually
every behavioral component involved in the four paradigms
described such as nociception, consummatory behavior
[124], social [67] and motor behavior. It is not, therefore,
easy to separate the effects of drugs acting at opiate receptors on anxiety from the other actions.
It has long been held that exogenous opiate agonists such
as morphine and heroin somehow reduce human anxiety.
However, the extent to which such resembles a BZ-like effect on clinical anxiety as against clearly non-pathological
anticipation of relief from pain, or, in opiate-dependent persons, withdrawal symptomatology, would promise to be the
subject of a potentially interminable debate. For this review
it is more appropriate, therefore, to examine opiate effects
on conflict behavior. Morphine was initially used in the punished operant behavior procedure as a control for possible
drug effects on nociception and no enhancement was reported [77]. Subsequently apparent anti-conflict effects of
exogenous opiate agonists have been found using punished
operant [117] and consummatory [186] behavior and social
interaction [72]. Opiates have uniformly proved less efficacious than BZs in this respect, though. However, taken together with a number of studies showing apparent anticonflict effects of opiate agonists in non-standard paradigms
[14, 94, 125], there is certainly some evidence of attenuation
of conflict behavior with these drugs.
There are analogous problems in the interpretation of the
effects of opiate antagonists on conflict behavior since most
studies to date either fail to control for other motivational
effects, assess effects on behavioral baselines which are already so low that further suppression is unlikely or study
opiate antagonists only in interaction with BZs (see below).
Only a single study demonstrates proconflict actions of
naloxone convincingly, by showing a specific increase in
food neophobia at a dose which was found to be too low to
affect appetite in a comparable test [59].
Although opioid peptides do not seem to be directly involved in the BZ receptor system, the possibility of their
involvement in the pharmacological effects of BZ is
suggested by the changes in opioid peptide concentrations
brought about by diazepam administration [63, 64, 193]. Interestingly, hypothalamic concentrations are increased,
whilst those in the corpus striatum are reduced with opioid
peptide concentrations in the other brain regions studied remaining unchanged. The involvement of GABA in such
changes is suggested both by the capacity of muscimol and
amino-oxyacetic acid to simulate this effect of diazepam and
the antagonism of this effect of diazepam by bicuculline [64].
Intuitively, it might be expected that these BZ/opiate interactions would be most important in the abuse and dependence liability of BZs [40,134] and perhaps their effects
on feeding and drinking where there is a large and growing
literature implicating opioid peptide involvement per se [40,
41, 124]. However, several studies bear upon the possible
involvement of opioid peptides in the anti-conflict effects of
BZs and these are surveyed here.
The opiate antagonist naloxone (20-100 mg/kg) has been

456

SHEPHARD

reported to antagonise the anti-conflict effects of chlordiazepoxide [11]. However, such naloxone doses are so high
relative to those which produce antagonism at opiate receptors and well known effects on nociception and consummatory behavior, that the specificity and significance of this finding are questionable and indeed the authors of this study
suggested that G A B A antagonist properties of naloxone may
be responsible for this antagonism [11]. In more moderate
doses, naloxone inhibits the enhancement of punished operant behavior produced by diazepam [62,90] though since
naloxone alone inhibits feeding [124], and suppresses behavior in this paradigm [168], possibly due to enhanced pain
sensitivity (e.g., [74,148]), the specificity of this action is also
debatable. The antineophobic actions of diazepam in both
rats [168] and hamsters [12] may also be antagonised by
naloxone, though another study reported no antagonism of
diazepam effects on neophobia with naloxone, but antagonism of enhanced home-cage feeding only [26]. This report
contrasts with the apparent selective action of naloxone on
food neophobia as opposed to appetite noted above.
Naloxone also failed to antagonise the effects of diazepam on
conditioned suppression [168]. Summing up, the behavioral
evidence implying a role for opioid peptides in anti-conflict
actions of BZs is hardly compelling. It is distinctly unfortunate that no study has satisfactorily ruled out intrinsic effects
of naloxone on the relevant behaviors and that virtually all
studies, behavioral [12, 62, 168, 172] and otherwise [63, 64,
193], suggesting BZ/opioid interactions have used diazepam
and therefore generalizations to all BZs would be unwise. It
would also be interesting to examine interactions of BZs and
their antagonists with endogenous or exogenous opiate
agonists.
CONCLUSION
The BZ receptor system itself is evidently a complex one
and the widely-presumed endogenous ligand which may act
physiologically at these sites has resisted elucidation. Identification of this substance or substances would, of course,
progress the study of the pharmacology and physiology of
anxiety considerably. However, differences in number or
affinity of BZ receptors do not readily explain variations in
emotional behavior or even responsivity to BZs on the rather
sparse evidence, for instance from genotypic differences, to
date. It therefore seems highly probable that other neurotransmitter systems are involved in the actions of BZs, and
they certainly have a role in the physiology of anxiety. This
review has sought to focus critically on the behavioral evidence for a role of neurotransmitters in anxiety and in the
mechanism of action of the BZs, viewed as far as possible as
separate questions. Attention has also been concentrated
upon what seem to be the major candidates in these contexts; serotonin, G A B A and the opioid peptides.
In relation to serotonin, the paucity of evidence implicating it in BZ action should not be allowed to obscure the large,
and relatively uncontroversial literature which suggests that
it has a role in anxiety. It probably also affects other motivational systems [17] but the implications of these for the
analysis of conflict behavior are unclear (see earlier section
on behavioral paradigms). The G A B A hypotheses of anxiety
and BZ action have to be regarded as the leading ones in this
area and the notion of a BZ/GABA receptor complex is beginning to receive behavioral support. However, there are a
number of anomalous findings and incomplete arguments in
behavioral research here. In particular, more research on the

effects of G A B A agonists on conflict behavior and BZ action

seems indicated and such should probably include more
studies using intracerebral administrations in specific brain
regions as only valproate seems effective when given peripherally, and this drug has an unclear mechanism of action. It
would also be interesting to seem more analysis of the reversibility of actions of GABAergic drugs by BZ antagonists,
and such methods could also be applied to other hypotheses
with advantage. The possible involvement of opioid peptides
in anxiety and anti-conflict effects of BZs are hypotheses
which can only be regarded as being in early stages of development at present. It is regrettable that only a limited range
of drugs have been used in their formation and that the opiate
agonists and antagonists used, typically morphine and
naloxone, have relatively non-specific effects at the various
opiate receptor sub-types [124]. Even if morphine-like opiate
agonists were convincingly shown to possess anxiolytic
properties, it is obvious that they could never be used clinically for this purpose. Again, further research with more
specific compounds, which may have reduced abuse liability
and taking care to use appropriate conflict paradigms seems
indicated.
Theoretically, the serotonin, GABA, opioid and BZ receptor systems could each have entirely independent actions
on brain anxiety systems. Although intuitively this might
seem unlikely, it should be noted that studies showing an
intermediate behavioral effect of a combination of drugs,
each of which alone produce opposite behavioral effects,
furnish no evidence that the drugs are acting at the same or
related receptor sites. Thus, such results do not refute the
notion of independent actions of two or more neurochemical
systems on anxiety, even though they are often loosely referred to as 'antagonism.' However, a number of behavioral
studies, together with our growing knowledge of the neural
circuitry of anxiety-related systems, enable at least informed
speculation as to how these neurochemical systems might be
interrelated, a type of analysis which complements
neuroanatomical models [86, 87, 100]
Regarding serotonin, although manipulations of this neurotransmitter system clearly affect conflict behavior and anxiety, such treatments do not modulate BZ action [106, 154.
159, 181]. Indeed, serotonergic drugs have little or no effect
on conflict behavior in the presence of BZs [159,181]. This
suggests that the site of action of BZs is distal or more fundamental to conflict processes than that of serotonergic
agents. This hypothesis is supported by consideration of the
anatomy of brain serotonin system which contains ascending
projections from the ventral raph6 to probable sites of the
anxiolytic action of BZs in such regions as the amygdala,
cortex and hippocampus [100]. Therefore, in the presence of
BZs activating their receptors at these latter sites, the functional importance of the serotonergic input is minimised.
Conversely, insofar as opioid systems have been shown to
have a role in conflict behavior per se and BZ action, such
evidence as there is would suggest that changes in opioid
systems follow activation of BZ receptors [63, 64, 193] and
that indirect activation of opiate receptors may be required
for anti-conflict effects of BZs to occur [12, 62, 90, 168].
Taken at face value, this would suggest that just as the actions of BZs on anxiety systems are more fundamental than
those of serotonergic drugs, in that the latter do not affect
conflict behavior in the presence of BZs, the opioid peptide
system may be an even more appropriate point at which to
produce pharmacological anxiolysis. The several cautions
raised earlier must temper this, however, and the anatomical

TRANSMITTERS, ANXIETY AND BENZODIAZEPINES

connections between BZs and opioid systems are largely unknown, except insofar as opioid peptide concentrations in
the corpus striatum and hypothalamus seem most sensitive
to modification by BZs. Without again rehearsing the evidence, it seems that BZs and G A B A act at different sites on a
common receptor complex and thus no interneuronal structure should be postulated. Therefore, although one can look
separately at the role of serotonin, G A B A and BZ receptor
systems, and of the possible involvement of opioid systems,
in conflict and anxiety, there is considerable and growing
evidence of interrelationships and knowledge of how these
comprise a physiological system.
The insistence of this review on examining the behavioral
pharmacology of established conflict paradigms is not intended to imply that other techniques, including other behavioral ones, are of little value. Regarding the latter the method
of drug discrimination, in which subjects are required to
learn one response following control injections and a different one following a test drug, has shown itself to be highly
sensitive and specific and most useful in clarifying mechanisms of drug action, for example, of hallucinogens [107].
This powerful technique is beginning to be applied to BZ
research on an appreciable scale [147] and might be expected
to be particularly useful in comparing possible endogenous
BZ receptor ligands to BZs themselves. BZs have also been

457

shown to antagonise the discriminative stimulus properties

of pentylenetetrazol, which has anxiogenic properties in humans [108]. However, some caution in adopting this procedure for the behavioral assessment of anxiolytics seems indicated since it has not been shown directly that the discriminative stimulus effects of pentylenetetrazol depend
upon anxiogenesis and BZs might not specifically antagonise
the stimulus properties, but rather produce a different internal state from either pentylenetetrazol alone or saline. Even
if these points could be answered convincingly, this procedure might not prove to be a suitable test for new anxiolytics,
since it might only detect those with a highly specific mechanism of action.
The use of selected strains of animals and their progeny
provides a non-intrusive means of manipulating emotional
behavior (e.g., [27,65]), BZ receptors [82, 144, 145, 162,
163], and behavioral responsivity to BZs [157,162] and thus
far the neurochemical parameters which must underlie such
relationships have received only superficial analysis [162].
What seems certain is that the next few years will see a
wealth of new findings in the general area surveyed here.
Behavioral pharmacologists must not allow themselves to be
bewildered by this, but must use the data to generate predictions which are testable in appropriate behavioral paradigms.

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