Psychopharmacology (1985) 85:143-147

Psychopharmacology
9 Spri nger-Verl ag 1985
f l - Carbol i nes can enhance or ant agoni ze the effects
of puni s hment in mi ce
D. N. Stephens and W. Kehr
Research Laboratories of Schering AG, 1000 Berlin 65 and Bergkamen, FRG
Abstract. Six fi-carboline ligands at central benzodi azepi ne
(BZ) recept ors were t est ed for t hei r anxiolytic or anxio-
genic propert i es in mice in t he four-pl at e test. ZK 93 423
and ZK 91 296 increased activity which had been sup-
pressed by puni shment (1 mA, 60 ms footshock) at doses
which exert ed no effect on unpuni shed l ocomot i on.
ZK 93 426, ZK 90 886, FG 7142, and DMCM exert ed no
antipunishment activity themselves, and antagonized t he
ability of diazepam to increase bot h punished and unpun-
ished l ocomot or activity. DMCM, FG7142, and
ZK 90 886, but not ZK 93 426, also enhanced t he ability of
a reduced level of foot shock (0.3 mA) to suppress activity.
This propuni shment activity of DMCM and ZK 90 886 t ook
place at doses which had no effect on unpunished
l ocomot i on. The nat ure of t he effect of the individual
fl-carbolines on puni shment was rel at ed to t he nat ure of
their i nt eract i on with t he BZ/ GABA recept or/ chl ori de
channel compl ex ( GBC complex). Thus t he antipunishment
propert i es of ZK 93 423 and ZK 91 296 were associated
with t hei r ability to increase binding of 35S-t-butylbicyclo-
phosphor ot hi onat e (TBPS) to its binding site associated
with t he chloride channel, whereas DMCM, FG 7142 and
ZK 90 886, which exert ed propuni shment effects, reduced
TBPS binding. ZK 93 426, which was neut ral with respect
to punished activity, had t he weakest effect on TBPS
binding. These results are discussed in the cont ext of a
possible role of t he GBC compl ex in anxiety.
Key words: fl-Carbolines - Anxi et y - Passive Avoi dance
- Mouse - DMCM - FG 7142 - ZK 90 886 - ZK 93 426
- ZK 91 296 - ZK 93 423
Ligands at central benzodi azepi ne (BZ) recept ors have
been classified into t hree overlapping groups, agonists,
which resembl e t he convent i onal BZs, antagonists, which
antagonize t he pharmacological action of the agonists, and
a third group, inverse agonists, which exhibit pharmaco-
logical propert i es opposite to those of conventional BZs
(Polc et al. 1982; Braest rup et al. 1982). The group of
compounds based on t he fl-carboline skeleton contains
several substances with a high affinity for BZ receptors.
Al t hough bot h t he fl-carboline, fl-carboline-3-carboxylic
acid ethyl est er (fl-CCE), which was originally isolated as an
artefact from human urine (Braest rup et al. 1980), and its
first derivatives, exhibited only inverse agonist propert i es
Offprint requests to: D. N. Stephens, D. Neuropsychopharmaco-
logy, Schering AG, Postfach 65 03 11, 1000 Berlin 65
(Braest rup et al. 1982), more recent l y/ 3-carbol i nes bot h
with agonist (Pet ersen et al. 1984) and antagonist (Jensen et
al. 1984) propert i es have been described. The availability of
such a group of chemically rel at ed compounds which
achieve t hei r het erogeneous pharmacological effects by
acting at a common r ecept or or set of recept ors provides a
useful t ool for investigating t he relationship of behavioural
effect to r ecept or events.
Convent i onal benzodiazepines are renowned for their
anxiolytic propert i es. The i nformat i on present l y available
on t he activities of t he fl-carbolines in models of anxiety is
limited. The antagonism of t he antipunishment propert i es
of phenobarbi t al by fl-CCE and by fi-carboline-3-carboxylic
acid met hyl amide ( FG 7142) has been cited as evidence for
an anxiogenic activity of these fl-carbolines (Pet ersen et al.
1982), and FG 7142 has been r epor t ed to induce intense
attacks of anxiety in human volunteers ( Dor ow et al. 1983).
Bot h fl-CCE and FG 7142 exert a proconflict action in
rodent s (Corda et al. 1983; de Carvalho 1983; Pet ersen et
al. 1983). fl-CCE has also been r epor t ed to give rise to an
anxiet~y-like syndrome in monkeys (Ninan et al. 1983). We
ourselves have r epor t ed that in rats trained to discriminate
bet ween anxiogenic doses of pent yl enet et razol and saline,
FG 7142 and t he convulsant 6,7-dimethoxy-4-ethyl-fl-car-
boline-3-carboxylic acid met hyl est er (DMCM) are iden-
tified as PTZ-l i ke, which we take to suggest that these
substances are also anxiogenic (Stephens et al. 1984a, b).
fl-CCM is also recognised as PTZ-l i ke (Lal and
Emmet t -Ogl esby 1984). 5-benzyloxy- ( ZK91296) and
6-benzyloxy- ( ZK 93 423) 4-methoxymethyl-fl-carbo-
line-3-carboxylic acid ethyl esters antagonized the PTZ cue,
suggesting t hey have anxiolytic propert i es, while 5-isopro-
poxy-4-methyl-fl-carboline-3-carboxylic acid ethyl ester
( ZK 93 426) was inactive in this test (Stephens et al.
1984a, b). In contrast, in the lick-suppression conflict test
also employing rats, ZK 93 426 has been r epor t ed to exhibit
proconflict activity (Jensen et al. 1984). "Anxi ogeni c"
actions of t he fl-carbolines fl-CCE and FG 7142 have also
been observed in t he rat social i nt eract i on test (File et al.
1982, File and Pellow 1984).
In the present study, we ext end our observations on t he
fl-carbolines to t hei r activity in mice in a modified version of
t he four-plate test (Boissier et al. 1968). Activity of BZ
recept or agonists in this test is highly correl at ed with their
affinity for BZ recept ors (Stephens et al. 1984c) but no
i nformat i on is yet available on t he activity of either BZ
r ecept or antagonists or inverse agonists. We have t her ef or e
tested t he ability of selected fl-carbolines, BZ agonists,
antagonists and inverse agonists, to reinstate l ocomot or
144
activity suppressed by punishment, to antagonize the
antipunishment activity of the benzodiazepine diazepam,
and to enhance the punishing properties of reduced levels
of shock.
Met hods
A modification of the four-plate test (Boissier et al. 1968)
was used. Naive NMRI mice of either sex, supplied by the
Department Tierzucht und -haltung, Schering AG, and
weighing 25 + 2 g were placed individually in the centre of a
rectangular chamber (23 x 18 x 30 cm high) whose floor
was divided into four equally sized metal plates. Following
20 s in which the mouse was allowed to explore freely, it
received a mild (see below) and brief (60 ms) electric shock
each time it crossed from one plate to another. The number
of such crossings in a 1-min period were taken as a measure
of exploratory activity. As a control for sedative or
stimulant effects, the effect of test substances on non-pun-
ished crossings was assessed in independent groups. In both
punished and unpunished conditions eight mice were tested
per drug dose.
Three types of test were carried out. In tests of
anxiolytic activity the mice were injected IP with test
substance suspended in 10% cremophor EL (CEL) in
saline, at a dose volume of approximately 0.2 ml/mouse.
Drugs were injected 30 rain before the test and a shock
level of 1 mA was employed. An identical procedure was
used for tests of anxiogenic activity, except that a shock
level of 0. 3mA was used. In tests of the ability of
fl-carbolines to antagonize the antipunishment properties of
benzodiazepines, a shock level of 1 mA was used./?-Car-
bolines were administered IP 30 min, and a standard 1.56
mg/kg dose of diazepam in CEL administered IP 15 min
before the test. (This dose of diazepam is twice the
minimally effective dose in this test, and gives rise to
occupation of about 50% of central benzodiazepine
receptors.) In tests of the ability of ZK 93 426 to antagonize
the propunishment activity of 3.13 mg/kg DMCM, a shock
level of 0.3 mA was used; ZK 93 426 was administered
30 min and DMCM 15 min before the test. Effects of test
fl-carbolines were assessed by comparison with
CEL-treated controls or appropriate diazepam- or
DMCM-treated groups using Kruskal-Wallis nonparamet-
ric tests of significance (Siegel 1956). Potencies were
estimated as the lowest doses (minimal effective dose,
MED) which gave rise to a significant effect (P < 0.05)
relative to the appropriate CEL-, diazepam- or
DMCM-treated control group.
Drugs. ZK 93 423, ZK 93 426 (both synthesized by Dr. D.
Seidelmann, Schering AG), ZK91296, DMCM (both
synthesized by Dr. G. Neef, Schering AG), ZK 90 886
(synthesized by Dr. U. Eder, Schering AG), FG 7142; AS
Ferrosan, Copenhagen, Denmark) and diazepam (Hoff-
mann-LaRoche, Basel, Switzerland) were dispersed by
ultrasound sonification in 0.9% saline solution containing
10% CEL.
Table 1. Comparison of the pro- or antipunishment properties of six fi-carbolines with their biochemical actions at the GBC
complex
Type of action MED Displacement of GABA a TBPS b % BZ receptorJ
in four plate test (mg/kg) 3H-flunitrazepam ratio ratio occupation
in vivo (EDs0, (rat) at MED
mg/kg, IP (mouse)
ZK 93 423 Antipunishment 3.1 1.0 c 2.17 c 1.58 f 76
ZK 91 296 Antipunishment 3.1 1.2 d 1.23 d 1.20 i 72
ZK 93 426 Antipunishment > 100 > 99
BZ-antagonism 4.0 0.3 e 1.37 e 0.88 i 93
Propunishment > 100 > 99
ZK 90 886 BZ-antagonism 6.25 3.0 f 1.18 f 0.86 f 68
Propunishment 1.25 29
FG 7 142 BZ-antagonism 25 10.0g 0.87 h 0.79 71
Propunishment 25 71
DMCM BZ-antagonism 12.5 3.1g 0.46 h 0.58 i 80
Propunishment 3.1 50
a Calculated as the ratio of the ICs0 s in the absence and in the presence of muscimol (50 ~tmol/1) for the displacement of 3H-diazepam in
vitro
b Calculated as the ratio of TBPS binding affinity to rat brain membranes in the absence and presence of BZ ligand concentrations giving
> 90% receptor occupation, using the method of Supavilai and Karobath (1983)
Unpublished values quoted in Braestrup et al. 1984
d Petersen et al. 1984
Jensen et al. 1984
Honore (unpublished)
g Jensen et al. 1983
h Braestrup et al. 1982
i Nielsen et al. 1984
J Calculated from EDs0 for the displacement of 3H-flunitrazepam binding in vivo using the relationship:
100
% occupation =
(EDs0/Dose) + 1
145
Re s u l t s
Fi gur e 1 shows t hat ZK 93 423 and ZK 91 296 si gni fi cant l y
i ncr eased expl or at or y crossi ngs t hat had been suppr essed
by puni s hment , but di d not i ncr ease unpuni s hed activity.
ZK 93 423 was nei t her par t i cul ar l y pot e nt nor effect i ve in
this t est ; at hi gher doses (25 mg/ kg) ZK 93 423 sever el y
r educed bot h unpuni s hed and puni s hed r espondi ng,
refl ect i ng its st r ong at axi c activity. DMCM, FG7 1 4 2 ,
ZK 90 886, and ZK 93 426 s howed no ability t o i ncr ease
puni s hed r es pondi ng at doses up t o 3. 1, 100, 30, and
30 mg/ kg respect i vel y. Hi ghe r doses of DMCM coul d not
be t est ed becaus e of its convul sant act i vi t y in mi ce at doses
above 5 mg/ kg ( Pet er s en 1983). The doses of FG 7142,
ZK 90 886, and ZK 93 426 whi ch wer e i neffect i ve gi ve
r ecept or occupanci es gr eat er t han 90% ( Tabl e 1).
Fi gur e 2 shows t hat t he f our /3-carbolines wi t hout
ant i puni s hment pr oper t i es ant agoni zed t he ability of
4 - p l a t e - t e s t
9 n u mb e r o f c r o s s i n g s
r
2 0 - ZK 9 1 2 9 6 ZK 9 3 4 2 3
1 2 - 1 i
84
4 0 - - - - 0 u n p u n i s h e d
_- _- p u n i s h e d
- - r ~ I I , , , , , , , ' I I - - ~ r - - r r - - - c r r -
e E L 0. 78 1. 56 3. 13 6, 25 12.5 2 5 CEL 0. 39 0 . 7 8 1 . 5 6 3 . 1 3 6 . 2 6 12.5 2 5
d r u g d o s e ( mg / k g i.p.)
Fig. 1. Numbers of crossings in the four-plate apparatus in
unpunished (open circles) or punished groups of mice (1 mA,
60 ms; closed circles) following treatment with vehicle or with ZK
93 423 or ZK 91 296. Bars indicate standard errors. * Different
from vehicle-treated control. P < 0.05, Kruskal-Wallis Test
1.56 mg/ kg di azepam, bot h t o r ei nst at e l oc omot or act i vi t y
suppr essed by shock and t o i ncr ease unpuni s hed act i vi t y in
t he novel t est envi r onment .
The f our di azepam ant agoni st s di f f er ed f r om each ot her
in t hei r pr opuni s hment act i vi t y when t est ed wi t h r educed
shock levels (0. 3 mA) . DMCM, FG 7142, and t o a weak
ext ent ZK 90 886 i ncr eased t he ability of mi l d f oot s hock t o
J3-carbolJne e n h a n c e me n t o f p u n i s h m e n t
(0.3 mA f o o t s h o c k )
9 n u mb e r o f c r o s s i n g s
DMCM FG 7 1 4 2
2 0 -
12-
8- b4~- , 3
4
L I 1 ' . 0 ' . 8 ' E L I I 6 ' ' ' '
Cl~ 0 2 3.1 C ! 25 2 5 100
ZK 9 0 8 8 6 ZK 9 3 4 2 6
2 0 -
4-
, II, LII ,
CEL 0.31 1)25 510 CE 2'.5 10 410
d r u g d o s e ( mg / k g i.p.}
Fig. 3. The ability of four fl-carbolines to enhance the effects of a
low shock level (0.3 mA, 60 ms) in suppressing locomotor activity
of mice in the four-plate apparatus. Open symbols indicate
unpunished and closed symbols punished groups. Bars indicate
standard errors. * Different from vehicle-treated group,
P < 0.05
9 n u mb e r o f c r o s s i n g s
1
4 - p l a t e - t e s t : A n t a g o n i s m o f D i e z e p a ~
n u mb e r o f c r o s s i n g s
DMCM 9 FG 7 1 4 2 * *
~
le di az epam 6. 25 2 5 100
J ~ ZK 9 3 4 2 6 ~ [ ] O u n s h o c k e d
[ [ ~ ~ ~ a z e p a m 4 16 3 2
1. 56 mg / k g d r u g d o s e ( mg / k g )
Fig. 2. Locomot or activity in unpunished (open symbols) and
punished (closed symbols) groups of mice. Bar diagrams indicate
the effects of vehicle and of a standard dose (1.56 mg/kg) of
diazepam. The ability of the fl-carboline to antagonise the effects
of diazepam are indicated by the open (unpunished) and closed
(punished) circles. Bars indicate standard errors. * Different from
vehicle-treated group, P < 0.05; + different from diaze-
pare-treated group, P < 0.05
1 6 -
1 4 -
1 2 -
8 - T
4- ~
0. 16 0. 63 2. 50 mg / k g
Z K 9 3 4 2 6
[2f u n p u n i s h e d - v e h i c l e
p u n i s h e d - v e h i c l e
g~ puni s hed- DMCM
Fig. 4. The ability of ZK 93 426 to antagonize the propunishment
properties of 3.13 mg/kg DMCM in mice punished with a low
shock level (0. 3mA, 60ms). Bars indicate standard errors.
* Different from punished-vehicle group, P < 0.05
146
suppress expl orat ory activity (Fig. 3). In t he case of DMCM
and ZK 90 886 this propuni shment effect occurred at doses
which did not reduce unpunished activity. FG 7142 had
similar effects on bot h punished and unpunished activity
(Fig. 3). In contrast, ZK 93 426 had no effect on ei t her
punished or unpuni shed activity. When given t oget her with
DMCM, ZK 93 426 antagonized t he propuni shment effects
of this substance (Fig. 4) (Kruskal Wallis H = 10.3; df = 3;
e < 0. 05) .
Di s c us s i o n
The six fl-carbolines t est ed for t hei r antipunishment,
propuni shment , or BZ antagonist activities in the present
experiments were chosen on t he basis of t hei r classification
as agonists, inverse agonists or antagonists at central BZ
recept ors, and their behavi our in t he four-plate tests closely
refl ect ed t he nat ure of their interactions with t he
GABA/ BZ recept or/ chl ori de channel complex ( GBC com-
plex). The full agonist ZK 93 423 exhibited antipunishment
activity, t hough t he powerful ataxic action of this substance
may have pr event ed expression of its full anxiolytic
potential in this test employing drug-naive animals.
ZK 91 296, which has been classified as a partial agonist
(Pet ersen et al. 1984) and does not exhibit ataxic proper-
ties, was also anxiolytic. ZK93426, a BZ recept or
antagonist (Jensen et al. 1984) exert ed nei t her pro- nor
antipunishment activity, but antagonised t he antipunish-
ment effect of diazepam and the propuni shment effect of
DMCM. The higher pot ency of ZK 93 426 in antagonising
t he propuni shment activity of DMCM compared to t he
antipunishment effects of diazepam can be explained by t he
fact that t he dose of DMCM used in this experi ment was the
MED for enhancing punishment, whereas the dose of
diazepam was twice the MED for antagonizing the effects
of punishment. The partial inverse agonists ZK 90 886 and
FG7142, as well as t he full inverse agonist DMCM
(Pet ersen 1983), all exhibited propuni shment activities.
Whet her t he action of FG 7142 in enhancing t he effect of
puni shment is truly an indication of a propuni shment
activity is difficult to j udge from t he present results, since
FG 7142 also reduced unpuni shed activity at t he same
doses. It may be wort h noting t hat in a lick suppression test,
a test of proconflict effects, Pet ersen and Jensen (1984)
found that FG 7142 l owered bot h punished and unpunished
licking, in contrast to fl-CCE, DMCM, and ZK 90 886
which reduced only punished licking. Lastly, like
ZK 93 426, FG 7142, ZK 90 886, and DMCM were all
effective in antagonising t he anxiolytic action of diaze-
pam.
Propuni shment actions of DMCM, FG7142, and
ZK 90 886 have previously been r epor t ed in conflict tests in
rodents (Corda et al. 1983; Pet ersen and Jensen 1984;
Pet ersen et al. 1983; de Carvalho et al. 1983) and we have
previously r epor t ed these t hree fi-carbolines are identified
as PTZ-like in rats t rai ned to discriminate the anxiogenic
cue provi ded by pent yl enet et razol (PTZ) (Stephens et al.
1984a, c).
In contrast to t he present observations, a propunish-
merit effect has also been attributed to ZK 93 426 (Jensen
et al. 1984), though nei t her an anxiolytic nor an anxio-
mimetic action of ZK93426 was seen in t he PTZ
discrimination test (Jensen et al. 1984; Stephens et al.
1984b). These apparent discrepancies may indicate little
mor e t han a differing sensitivity of the various tests. Thus,
the lick suppression test used by Jensen et al. (1984) seems
to be very sensitive to anxiomimetic substances while
perhaps being rat her insensitive to anxiolytics. ZK 91 296,
for instance, was inactive as an anxiolytic in the lick
suppression test (Pet ersen et al. 1984) but was active in t he
four-plate test (present report ), in a Geller conflict test
(Pet ersen et al. 1984), and partly active in antagonising t he
anxiety-like discriminative stimulus provi ded by PTZ
(Stephens et al. 1984a).
ZK 93 423, which was weakly anxiolytic in mice in t he
four-plate test, also shows anxiolytic activity in the PTZ
discrimination test, and in rats t rai ned to discriminate t he
benzodiazepine chlordiazepoxide from saline is identified
as chlordiazepoxide-like (Stephens et al. 1984a).
Bot h the anxiolytic and t he anxiogenic properties of the
various fl-carbolines appear to be medi at ed t hrough t he BZ
recept or. Thus, t he BZ- r ecept or antagonist Ro 15-1788
(Hunkel er et al. 1981) has been r epor t ed to antagonise t he
anxiolytic and anxiogenic effects of fl-carbolines in various
animal models (Corda et al. 1983; Kehr and Stephens 1984;
Ninan et al. 1982; Stephens et al. 1984a). Additionally, the
/3-carbolines DMCM, FG 7142, ZK 90 886, and ZK 93 426
all antagonize t he antipunishment activity of t he BZ,
diazepam (present experi ment ) as well as the ability of the
BZ, chlordiazepoxide, to provi de a discriminative stimulus
(Stephens et al. 1984a, and unpublished). Fur t her mor e,
Tabl e i indicates that whet her a particular fi-carboline
exhibits an anxiogenic, anxiolytic or neut ral activity is well
predi ct ed by t he nat ure of its interactions with t he
GABA/ BZ recept or/ chl ori de channel compl ex ( GBC com-
plex).
Thus, those BZ recept or ligands which increase t he
binding of GABA to its recept or, and whose own binding is
itself increased in t he presence of GABA agonists (Martin
and Candy 1978; Ehl ert et al. 1981) act as anxiolytics,
whereas those ligands which show a negative cooperativity
with GABA exhibit anxiogenic propert i es (Table 1; and
Stephens et al. 1984b). The GABAA recept or agonist
muscimol also possesses anxiolytic activity when adminis-
t ered centrally (Cananzi et al. 1980; Pet ersen and
Scheel-Kruger 1982; Scheel-Kruger and Pet ersen 1982) to
overcome its poor penet rat i on of t he brain. An exception to
t he relationship bet ween anxiolytic activity and effect on
GABA activity is provi ded by ZK 93 426, which in the
present experi ment s exhibited nei t her pro- nor antipunish-
ment propert i es, but whose affinity for the BZ recept or is
enhanced in t he presence of the GABA agonist muscimol
(Jensen et al. 1984).
The final out come of interactions with t he GBC
complex is an increase or decrease in conductance at t he
; c hl or i de channel. 35S-t-butylbicyclophosphorothionate
(TBPS) is t hought to specifically label this channel (Squires
et al. 1983) and a ratio of TBPS binding in t he presence and
absence of high concentrations of BZ- r ecept or ligand is
t hought t o reflect whet her t he ligand increases (agonists) or
decreases conduct ance (Supavilai and Karobat h 1983;
Nielsen et al. 1984). Tabl e 1 shows TBPS ratios for t he
fl-carbolines tested in the present experi ment and in this
case t he value of t he TBPS ratio predicts accurately the
anxiolytic, neut ral or anxiogenic propert i es of the six
fl-carbolines (see also Stephens et al. 1984c). Whet her t he
dissociation bet ween t he GABA ratio and TBPS ratio of
ZK 93 426 indicates t hat ZK 93 426 exerts a direct effect on
147
t he chl or i de channel i nst ead of, or i n addi t i on t o, an
i ndi r ect effect vi a t he B Z / GAB A r ecept or compl ex is not
clear.
I n s ummar y, t he pr es ent resul t s conf i r m t hat BZ
r ecept or l i gands can exer t ei t her anxi ol yt i c or anxi ogeni c
effect s and suggest t hat t he anxi ogeni c or anxi ol yt i c nat ur e
of such l i gands depends upon t hei r abi l i t y t o decr ease or
i ncrease conduct ance at t he chl or i de channel associ at ed
wi t h t he B Z / GAB A r ecept or compl ex. Ot he r c ompounds
whi ch modul a t e anxi et y ma y act di r ect l y upon t he chl or i de
channel .
Acknowledgements. We thank Tage Honore for allowing us to
quote his unpublished data on ZK 90 886 and Christel Schneider,
Ronald Weidmann and Monika Mfiller-Seewald for carrying out
the four-plate tests. Herbert Schneider and Theodora Duka
provided helpful comments on earlier drafts of the manu-
script.
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Received July 31, 1984; Final version October 23, 1984