0022-3565/02/3001-2–8$3.
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THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2002 by The American Society for Pharmacology and Experimental Therapeutics
JPET 300:2–8, 2002
Perspectives in Pharmacology
A New Benzodiazepine Pharmacology
H. MÖHLER, J. M. FRITSCHY, and U. RUDOLPH
Institute of Pharmacology and Toxicology, University of Zurich, Zurich, Switzerland (H.M., J.M.F., U.R.); and Department of Applied
Biosciences, Swiss Federal Institute of Technology, Zurich, Switzerland (H.M.)
Received March 5, 2001; accepted July 5, 2001 This paper is available online at http://jpet.aspetjournals.org
ABSTRACT
Classical benzodiazepine drugs are in wide clinical use as
anxiolytics, hypnotics, anticonvulsants, and muscle relaxants.
They act by enhancing the ␥-aminobutyric acidA (GABAA) re-
ceptor function in the central nervous system. The pharmaco-
logical relevance of the multitude of structurally diverse GABAA
receptor subtypes has only recently been identified. Based on
an in vivo point mutation strategy, ␣1-GABAA receptors were
found to mediate sedation, anterograde amnesia, and part of
GABAergic inhibition is one of the most rapidly developing
topics in neuropharmacology. New therapeutic opportunities
arise due to increasing insights into the molecular architec-
ture and diversity of the components involved in signal trans-
duction such as GABAA receptors, GABAB receptors, and
GABA transporters (Fig. 1). GABAA receptors are important
drug targets representing the sites of action of benzodiazepines,
barbiturates, and neurosteroids. The present article focuses on
the pharmacological distinction of GABAA receptor subtypes as
a basis for the development of new drugs that target restricted
neuronal networks. In particular, new ligands of the benzodi-
azepine site acting selectively on GABAA receptor subtypes are
expected to dissect the pharmacological spectrum of classical
benzodiazepines and display a minimum of side effects. For
further information on GABAA receptor subtypes other recent
reviews may be consulted (Barnard et al., 1998; Möhler et al.,
2000; Olsen and Homanics, 2000; Whiting et al., 2000; Möhler,
2001).
Synaptic Action of Benzodiazepines
At synapses, GABAA receptors are activated by a brief
nonequilibrium exposure to high concentrations of GABA.
Consistent with an increase in the affinity of the receptors for
GABA, therapeutically active benzodiazepines prolonged the
decay of spontaneous miniature inhibitory postsynaptic cur-
rents (mIPSC). Similarly, the amplitude of mIPSC was found
ABBREVIATIONS: GABA, ␥-aminobutyric acid; mIPSC, miniature inhibitory postsynaptic currents.
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the seizure protection, whereas ␣2-GABAA receptors, but not
␣3-receptors, mediate anxiolysis. Rational drug targeting to
specific receptor subtypes has now become possible. Only
restricted neuronal networks will be modulated by the new
subtype-selective drugs. Promising new anxiolytics have al-
ready been developed. A new pharmacology of the benzodiaz-
epine site is on the horizon.
to be enhanced by a benzodiazepine agonist in various neu-
ronal systems (Perrais and Ropert, 1999; Hajos et al., 2000),
suggesting that the drug-induced increase of the affinity for
GABA resulted in the recruitment of more receptors for ac-
tivation by GABA. However, in other neuronal systems the
amplitude of the mIPSC remained unaltered by a benzodiaz-
epine agonist (Mody et al., 1994; Poncer et al., 1996; Hajos et
al., 2000), which has been interpreted to indicate that the
release of a single quantum of GABA saturates all of the
available GABAA receptors in the respective synapses induc-
ing a maximal peak response without further enhancement
by the drug. Thus, at synapses that generate mIPSCs, the
postsynaptic receptor occupancy by GABA appears to be cell-
and synapse-specific, reflecting local differences in the num-
ber of receptors or the GABA concentration in the cleft.
Accordingly, the influence of benzodiazepine agonists on the
amplitude of mIPSCs appears to vary with the operational
configuration of the GABAergic synapse (Hajos et al., 2000).
In summary, the enhancement of a GABAergic inhibitory
response by a benzodiazepine agonist is based on the pro-
longed decay of the mIPSC and a potential increase of the
mIPSC amplitude. Even if the peak mIPSC amplitude is not
enhanced per se, the drug-induced prolongation of individual
mIPSCs will be reflected in an increased peak amplitude of
the compound inhibitory response caused by the summation
of several miniature currents (Mody et al., 1994).

Fig. 1. Scheme of a GABAergic synapse depicting the major elements of
signal transduction. GABAA receptors are indirectly linked to the synap-
tic anchoring protein gephyrin.
GABAA Receptors and Their Multiplicity
Based on the presence of 7 subunit families comprising at
least 18 subunits in the central nervous system (␣1– 6, ␤1–3,
␥1–3, ␦, ⑀, ␪, ␳1–3,) the GABAA receptors display an extraor-
dinary structural heterogeneity. Most GABAA receptor sub-
types in vivo are believed to be composed of ␣-, ␤-, and ␥-
subunits. The role of the ␦-, ⑀-, and ␪- subunits, which have a
very limited expression pattern in the brain, remains to be
determined, but it is possible that they substitute for the
␥-subunit in ␣-␤-␥ combinations. The physiological signifi-
cance of the structural diversity of GABAA receptors lies in
the provision of receptors that differ in their channel kinet-
ics, affinity for GABA, rate of desensitization, and subcellular
positioning. In addition, the GABAA receptor subtypes can be
distinguished by their pharmacology.
Synaptic and Extrasynaptic GABAA Receptors. The
first electron microscopic studies of GABAA receptors re-
vealed the ubiquitous presence of extrasynaptic GABAA re-
ceptors in the cerebellum, thalamus, and cerebral cortex
(Somogyi, 1989). In fact, synaptic GABAA receptors are best
seen using postembedding electron microscopy, or by immu-
nofluorescence staining using weakly fixed brain sections
(Fritschy et al., 1998a), showing pronounced enrichment in
the postsynaptic density of GABAergic synapses compared
with extrasynaptic sites (Nusser et al., 1995). These studies
also revealed differential targeting of GABAA receptor sub-
types to different types of synapses. For instance, the ␣2
subunit in hippocampal pyramidal cells is concentrated in
synapses on the axon-initial segment (Nusser et al., 1996a;
Fritschy et al., 1998a), as well as in synapses formed by
cholecystokinin-positive basket cells on the soma (Nyı́ri et
al., 2001). The ␣6 subunit, which represents diazepam-insen-
sitive GABAA receptors in the cerebellum can be found both
in excitatory and inhibitory synapses in glomeruli of the
granule cell layer, as well as extrasynaptically (Nusser et al.,
1996b,1999; Sassoè-Pognetto et al., 2000). Finally, GABAA
receptors containing the ␦-subunit in the cerebellum are ex-
clusively found at extrasynaptic sites (Nusser et al., 1998).
Both extrasynaptic receptor types mediate tonic inhibition of
neuronal activity (Brickley et al., 1996, 2001).
Benzodiazepine Pharmacology 3
Synaptic and extrasynaptic GABAA receptors differ in
their kinetic properties in line with their distinct functional
roles. For instance, extrasynaptic GABAA receptors contain-
ing the ␦-subunit in dentate gyrus and cerebellum are tailor-
made for tonic inhibition, due to their high affinity for GABA
and slow desensitization kinetics (Brickley et al., 1996; Mody
and Nusser, 2000). Marked differences in desensitization
kinetics have also been reported for synaptic and extrasyn-
aptic receptors in inferior olivary neurons. GABAA receptors
containing the ␣2-subunit are postsynaptic and characterized
by rapid desensitization kinetics, whereas extrasynaptic
GABAA receptor containing the ␣3-subunit desensitize very
slowly (Devor et al., 2001). Interestingly, such differences are
not evident in recombinant expression systems, suggesting
the contribution of additional factors regulating GABAA re-
ceptor function at synaptic and extrasynaptic sites. A further
identification of the molecular composition of GABAergic
postsynaptic densities is expected to shed light on the func-
tional regulation of synaptic GABAA receptors (Moss and
Smart, 2001).
The demonstration that gephyrin, a synaptic clustering
protein initially isolated with glycine receptors, is present in
a subset of GABAergic synapses in the retina (Sassoè-
Pognetto et al., 1995) prompted the analysis of its role in
relation to GABAA receptors. Gephyrin was shown to be
required, along with the ␥2-subunit, for postsynaptic cluster-
ing of major GABAA receptor subtypes (Essrich et al., 1998;
Kneussel et al., 1999). A recent study demonstrated by im-
munoelectron microscopy and double-immunofluorescence
staining that gephyrin is colocalized with the vast majority of
postsynaptic GABAA receptors throughout the central ner-
vous system (Sassoè-Pognetto et al., 2000), indicating that it
represents a useful marker of GABAergic (and glycinergic)
synapses.
Diazepam-Sensitive GABAA Receptors. Receptors con-
taining the ␣1-, ␣2-, ␣3-, or ␣5- subunits in combination with
any of the ␤-subunits and the ␥2-subunit are most prevalent
in the brain (Fig. 2). These receptors are sensitive to benzo-
diazepine modulation. The major receptor subtype is assem-
bled from the subunits ␣1␤2␥2, with only a few brain regions
lacking this receptor (granule cell layer of the olfactory bulb,
reticular nucleus of the thalamus, spinal cord motoneurons)
(Fritschy and Möhler, 1995; Pirker et al., 2000) (Table 1).
Receptors containing the ␣2- or ␣3- subunit are consider-
ably less abundant and are highly expressed in brain areas
where the ␣1-subunit is absent or present at low levels (Table
1). The ␣2- and ␣3-subunits are frequently coexpressed with
the ␤3- and ␥2-subunits, which is particularly evident in
hippocampal pyramidal neurons (␣2␤3␥2) and in cholinergic
neurons of the basal forebrain (␣3␤3␥2). The ligand-binding
profile of these receptors differs from that of ␣1␤2␥2 by having
a considerably lower displacing potency for ligands such as
3-carboxymethoxy-␤-carboline, CL 218,872, and zolpidem
(Table 1).
Receptors containing the ␣5-subunit are of minor abun-
dance in the whole brain (Table 1) but are expressed to a
significant extent in the hippocampus, where they comprise
15 to 20% of the diazepam-sensitive GABAA receptor popu-
lation, predominantly coassembled with the ␤3-and ␥2-sub-
units. The ␣5-receptors are differentiated from ␣1␤2␥2,
␣2␤3␥2, and ␣3␤3␥2 receptors by a lower affinity to CL
218,872 and near insensitivity to zolpidem (Table 1).
4 Möhler et al.
Fig. 2. The four classes of diazepam-sensitive GABAA receptors are distinguished by the type of ␣-subunit (␣1, ␣2, ␣3, ␣5) and their largely distinct
neuronal localizations as demonstrated immunohistochemically in mouse brain sections. The major known pharmacological actions mediated via the
respective receptor subtypes are indicated.
The ␥1- and ␥3-subunits characterize a small population of
receptors that contain various types of ␣- and ␤-subunits.
Due to their reduced affinity for the classical benzodiaz-
epines, they do not appear to contribute to any great extent to
their pharmacology in vivo (Möhler et al., 2000; Whiting et
al., 2000; Möhler, 2001).
Diazepam-Insensitive GABAA Receptors. GABAA re-
ceptors that do not respond to clinically used ligands, such as
diazepam, flunitrazepam, clonazepam, and zolpidem, are of
low abundance in the brain and are largely characterized by
the ␣4- and ␣6-subunits (Table 1). Receptors containing the
␣4-subunit are generally expressed at very low abundance
but more prominently in thalamus and dentate gyrus (Pirker
et al., 2000); those containing the ␣6-subunit are restricted to
the granule cell layer of the cerebellum (about 30% of all
GABAA receptors in the cerebellum). Both receptor popula-
tions are structurally heterogeneous, and the majority of the
␣6-containing receptors are of the ␣6␤2␥2 combination (Table
1). The benzodiazepine site profile of ␣4- and ␣6-receptors is
characterized by a low affinity for flumazenil and bretazenil
and a switch in the efficacy of Ro 15-4513 (ethyl 8-acido-5,6-
dihydro-5-methyl-6-oxo-4H-imidazol[1,5-␣] [1,4]benzodiaz-
epine-3-carboxylate) from an inverse agonist to an agonist.
The ␦-subunit is frequently coassembled with the ␣4- or the
␣6-subunit in benzodiazepine insensitive receptors (Möhler
et al., 2000; Whiting et al., 2000; Möhler, 2001).
In the retina, homomeric receptors consisting of the ␳-sub-
unit represent a particular class of GABA-gated chloride
channels. Their GABA site is insensitive to bicuculline and
baclofen, and they are not modulated by barbiturates or
benzodiazepines (Table 1). Due to these distinctive features
the receptors are frequently termed GABAc receptors (Bor-
mann, 2000), although they can also be considered as a
homomeric class of GABAA receptors (Barnard et al., 1998).
Distinct Benzodiazepine Sites Per Receptor
The benzodiazepine site is thought to be located at the
interface of the respective ␣-subunit (␣1, ␣2, ␣3, ␣5) and the
␥2-subunit. About 25% of ␣1-receptors in rat brain contain a
second type of ␣-subunit, i.e., the ␣1-subunit is coassembled
with the ␣3-subunit. If there is no preference as to which
subunit holds the position adjacent to the ␥2-subunit, a mixed
pharmacology for the benzodiazepine site would be expected.
Receptors with ␣1 and ␣3 subunits displayed a ligand-binding
profile characteristic of both ␣3 and ␣1 subunits with the
latter being predominant (Araujo et al., 1996). Therefore,
each ␣ subunit appears to contribute its own binding char-
acteristics. For ␣1␣6␤␥2 receptors, which represent about
40% of all ␣6 receptors (Pollard et al., 1995; Khan et al., 1996;
Jechlinger et al., 1998), conflicting results were reported with
either each ␣-subunit contributing its typical pharmacology
(Khan et al., 1996) or a single pharmacology typical for ␣6
(Pollard et al., 1995). Similarly, in radioligand binding assays
TABLE 1
GABAA Receptor Subtypes
Composition Pharmacological Characteristicsa
␣1␤2␥2 Major subtype (60% of all GABAA receptors). Mediates the
sedative, amnestic, and—to a large extent—the anticonvulsant
action of benzodiazepine site agonists. High affinity for classical
benzodiazepines, zolpidem, and the antagonist flumazenil.
␣2␤3␥2 Minor subtype (15–20%). Mediates anxiolytic action of
benzodiazepine site agonists. High affinity for classical
benzodiazepine agonists and the antagonist flumazenil.
Intermediate affinity for zolpidem.
␣3␤n␥2 Minor subtype (10–15%). High affinity for classical
benzodiazepine agonists, and the antagonist flumazenil.
Intermediate affinity for zolpidem
␣4␤n␥/␣4␤n␦ Less than 5% of all receptors. Insensitive to classical
benzodiazepine agonists and zolpidem.
␣5␤1/3␥2 Less than 5% of all receptors; high affinity for classical
benzodiazepine agonists and the antagonist flumazenil. Very
low affinity for zolpidem.
␣6␤2,3␥2 Less than 5% of all receptors. Insensitive to classical
benzodiazepine agonists and zolpidem.
␣6␤n␦ Minor population. Lacks benzodiazepine site.
␳ Homomeric receptors: insensitive to bicuculline, barbiturates,
baclofen, and all benzodiazepine site ligands. Also termed
GABAc receptor; for nomenclature see Barnard et al., 1998.
a
Reviewed in Macdonald and Olsen, 1994; Barnard et al., 1998; Rudolph et al., 1999; Löw et al., 2000; Möhler et al., 2000; Whiting et al., 2000; Möhler, 2001. The term classical benzodiazepines refers to diazepam and
structurally related agonists in clinical use.
b
Synaptic localization is based mainly on ultrastructural evidence or on the colocalization with gephyrin and refers to the respective type of ␣-subunit.
Neuronal Localizationb
Synaptic and Cerebral cortex (Somogyi, 1989; J.M. Fritschy, unpublished data)
extrasynaptic Hippocampus, dentate gyrus (interneurons and principal cells) (Nusser et al., 1995)
Pallidum (Somogyi et al., 1996)
Striatum (interneurons) (J.M. Fritschy, unpublished data)
Thalamic relay nuclei (Somogyi, 1989; J.M. Fritschy, unpublished data)
Olfactory bulb (mitral cells and interneurons) (Giustetto et al., 1998)
Cerebellum (Purkinje cells, stellate, basket cells, and granule cells) (Somogyi et al., 1996;
Nusser et al., 1997, 1998, 1999; Sassoè-Pognetto et al., 2000)
Deep cerebellar nuclei (Sassoè-Pognetto et al., 2000)
Synaptic Cerebral cortex (Fritschy et al., 1998a)
Hippocampus, dentate gyrus (principal cells mainly on the axon initial segment) (Sassoè-
Pognetto et al., 2000; Fritschy et al., 1998a; Nusser et al., 1996a)
Olfactory bulb (granule cells) (Sassoè-Pognetto et al., 2000)
Striatum (spiny stellate cells) (Fritschy et al., 1998a)
Inferior olivary neurons (mainly on dendrites) (Devor et al., 2001)
Synaptic Cerebral cortex (principal cells in particular in layers V and VI; some axon initial
segments) (Fritschy et al., 1998a)
Hippocampus (some hilar cells) (J.M. Fritschy, unpublished data)
Olfactory bulb (tufted cells) (Giustetto et al., 1998)
Thalamic reticular nucleus (Fritschy et al., 1998a)
Cerebellum (Golgi cells) (Sassoè-Pognetto et al., 2000)
Medullary reticular formation (Fritschy et al., 1998a)
Extrasynaptic Inferior olivary neurons (Devor et al., 2001)
Extrasynaptic Dentate gyrus (granule cells) (Mody and Nusser, 2000)
Synaptic Spinal trigeminal nucleus, superior olivary neurons (J.M. Fritschy, unpublished data)
Extrasynaptic Cerebral cortex (J.M. Fritschy, unpublished data)
Hippocampus (pyramidal cells) (Fritschy et al., 1998b)
Olfactory bulb (granule cells) (Fritschy et al., 1998b)
Synaptic Cerebellum (granule cells) (Nusser et al., 1996b, 1998, 1999)
Extrasynaptic Cerebellum (granule cells) (Nusser et al., 1996b, 1998, 1999; Sassoè-Pognetto et al., 2000)
Synaptic Retina (Kaulen et al., 1998)
Benzodiazepine Pharmacology
5
6 Möhler et al.
of hippocampal ␣5 receptors, only the ␣5 pharmacology was
apparent (Araujo et al., 1999), although the receptors contain
an additional ␣1-, ␣2-, or ␣3-subunit (Sur et al., 1998; Araujo
et al., 1999). Thus, the impact of distinct ␣-subunits on the
receptor pharmacology remains to be defined.
Recently, a novel, low affinity benzodiazepine site was
identified on recombinant GABAA receptors (␣1␤2␥2)
(Walters et al., 2000). It displayed micromolar affinity for
diazepam and was insensitive to flumazenil. This site is not
of therapeutic relevance since practically all clinically rele-
vant effects of benzodiazepine drugs can be blocked by fluma-
zenil. In addition, the presence of the ␥2-subunit was not a
prerequisite for the low affinity site since it is also present on
␣1␤1-receptors (Walters et al., 2000). At present it cannot be
excluded that the low-affinity site represents the so-called
peripheral benzodiazepine site, which is known to occur also
on GABAA receptors (Haefely, 1994).
Pharmacology of GABAA Receptor Subtypes
in Vivo
In the search for benzodiazepine site ligands with higher
therapeutic selectivity and a reduced side effect profile,
GABAA receptor subtypes have long been considered to be
promising targets. However, it was only recently that the
pharmacological relevance of GABAA receptor subtypes was
identified based on a gene knock-in strategy (Rudolph et al.,
1999; Löw et al., 2000). The benzodiazepine site was ren-
dered diazepam-insensitive by a point mutation in the re-
spective ␣ subunits by replacing a histidine residue with an
arginine residue [␣1(H101R), ␣2(H101R), ␣3(H126R), and
␣5(H105R)] (Wieland et al., 1992; Benson et al., 1998). Mouse
lines were generated in which the ␣1-, ␣2-, or ␣3-receptor
subtypes were diazepam-insensitive. In these mice certain
benzodiazepine effects were expected to be blunted, which
was attributed to the respective point-mutated receptor. This
strategy permitted the allocation of the benzodiazepine drug
actions to identified GABAA receptor subtypes. In addition, it
implicated the neuronal networks expressing the particular
receptor in mediating the corresponding drug actions.
The ␣1-, ␣2-, and ␣3-point-mutated receptors displayed a
level of expression and a regional and cellular distribution
that was indistinguishable from those in wild-type mice. In
addition, the gating of the point-mutated receptors by GABA
remained unaltered. Furthermore, the neomycin resistance
marker introduced by the replacement vector was eliminated
from the genome of all mouse lines generated by cre-loxP-
mediated recombination. The pharmacological analysis of the
point-mutated mice was therefore free of any potential inter-
ference, which may have resulted from the presence of the
neomycin resistance marker. Thus, a deficit in the behavioral
response to diazepam in the point-mutated mouse lines is
attributed to the pharmacological role of the respective re-
ceptor subtype (Rudolph et al., 1999; Löw et al., 2000).
Receptors Mediating Sedation. Sedation is a major
property of many benzodiazepine site ligands and has now
been shown to be mediated via ␣1-receptors. Among ␣1-, ␣2-,
and ␣3-point-mutated mice only the ␣1(H101R) mutants were
resistant to the depression of motor activity by diazepam and
zolpidem (Rudolph et al., 1999; Crestani et al., 2000a; Löw et
al., 2000). This effect was specific for ligands of the benzodi-
azepine site since pentobarbital or a neurosteroid remained
as effective in ␣1(H101R) mice as in wild-type mice in reduc-
ing motor activity. An ␣1(H101R) mouse line was also gener-
ated by McKernan et al.(2000). When measured under nov-
elty (“stress”) conditions, diazepam increased locomotion
compared with wild-type (McKernan et al., 2000). This effect
is apparently dependent on the test procedure and can be
induced also in the ␣1(H101R) mice generated by Rudolph et
al. (1999) under comparable test conditions (Crestani et al.,
2000b).
Receptors Mediating Amnesia. Anterograde amnesia is
a classical side effect of benzodiazepine drugs. The memory-
impairing effect of diazepam, analyzed in a step-through
passive avoidance paradigm, was strongly reduced in the
␣1(H101R) mice compared with wild-type mice as shown by
the increased latency for reentering the dark compartment
24 h after training (Rudolph et al., 1999). This effect was not
due to a potential nonspecific impairment since the ability of
a muscarinic antagonist to induce amnesia was retained in
the ␣1(H101R) mice. These results demonstrate that the di-
azepam-induced anterograde amnesia is mediated by ␣1-re-
ceptors.
Receptors Providing Protection Against Seizures.
The anticonvulsant activity of diazepam, assessed by its pro-
tection against pentylenetetrazole-induced tonic convulsions,
was reduced in ␣1(H101R) mice compared with wild-type
mice (Rudolph et al., 1999). The anticonvulsant effect of
diazepam, which remained in the ␣1(H101R) mice, was due to
GABAA receptors other than ␣1, since it was antagonized by
flumazenil. Sodium phenobarbital remained fully effective as
an anticonvulsant in ␣1(H101R) mice with a dose response
intensity similar to that of wild-type mice. These results
show that the anticonvulsant activity of benzodiazepines is
partially but not fully mediated by ␣1-receptors. The anticon-
vulsant action of zolpidem is exclusively mediated by ␣1-
receptors, since its anticonvulsant action is completely ab-
sent in ␣1(H101R) mice (Crestani et al., 2000a).
Receptors for Anxiolysis. New strategies for the devel-
opment of daytime anxiolytics that are devoid of drowsiness
are of high priority. The particular receptor subtype that
mediates the anxiolytic activity of benzodiazepine drugs was
recently identified (Löw et al., 2000). When the reactivity to
naturally aversive stimuli is measured in wild-type mice,
diazepam increases the time spent in the lit area of the
light/dark choice test and on the open arms of the elevated
plus maze, respectively. In contrast, the ␣2-point-mutated
mice were resistant to the effect of diazepam in these test
paradigms (Löw et al., 2000). This lack of response was
specific for ligands of the benzodiazepine site since
␣2(H101R) mice retained the ability to display an anxiolytic-
like response to sodium phenobarbital. Thus, the anxiolytic-
like action of diazepam is selectively mediated by the en-
hancement of GABAergic transmission in a population of
neurons expressing the ␣2-GABAA receptors. Thus, the ␣2-
GABAA receptors are highly specific targets for the develop-
ment of future selective anxiolytic drugs. They represent only
about 15% of all diazepam-sensitive GABAA receptors. Selec-
tive ligands are therefore expected to be devoid of the major
side effects that afflict the classical benzodiazepine anxiolyt-
ics.
It had previously been assumed that the anxiolytic action
of diazepam is based on the dampening of the reticular acti-
vating system. It is mainly represented by noradrenergic and

serotonergic neurons of the brain stem, which express exclu-
sively ␣3-receptors. The analysis of the ␣3-point-mutated
mice [␣3(H126R)] indicated that the anxiolytic effect of ben-
zodiazepine drugs, measured as described above, is not me-
diated by ␣3-receptors (Löw et al., 2000). The reticular acti-
vating system therefore does not appear to be a major
contributor to anxiolysis. In contrast, the ␣2-GABAA recep-
tors are highly expressed in cells in the cerebral cortex and
the hippocampus, including their pyramidal cells, which dis-
play particularly high densities of ␣2-GABAA receptors on
their axon initial segment (Nusser et al., 1996a; Fritschy et
al., 1998a). Thus, controlling the output of these principal
neurons may contribute to anxiolysis.
Receptors Mediating Myorelaxation. The degree of
muscle tone can be assessed in the horizontal wire test, in
which the ability of the animals to grasp and hang onto a
wire is measured. The muscle relaxant effect of diazepam is
largely mediated by ␣2-GABAA receptors, as shown by the
failure of diazepam to induce changes in muscle tone in the
␣2-mutated mouse line (Crestani et al., 2001). It was only at
high doses that ␣3-receptors were also implicated. Besides
the limbic system (see above), ␣2-receptors are highly specif-
ically expressed in the spinal cord, notably in the superficial
layer of the dorsal horn and in motor neurons (Bohlhalter et
al., 1996) with the latter being most clearly implicated in
muscle relaxation. It is important to note that the muscle
relaxant effect requires higher doses of diazepam than its
anxiolytic activity. This is attributed to a higher receptor
occupancy required for muscle relaxation.
Novel Subtype-Selective Benzodiazepine Site
Ligands
Among the clinically used ligands of the benzodiazepine
site only the hypnotic zolpidem displays a pronounced pref-
erential subtype selectivity (Langer et al., 1992) (Table 1).
Additional benzodiazepine site ligands with subtype selectiv-
ity are under experimental or clinical investigation (Hood et
al., 2000) and include the following agents.
L-838,417. The benzodiazepine site ligand L-838,417 in-
teracts with comparable affinity with ␣1-, ␣2-, and ␣3-recep-
tors and with only 3-fold lower affinity with ␣5-receptors.
However, it displays a dramatic subtype-selective efficacy.
L-838,417 fails to modulate the GABA response at ␣1-recep-
tors but enhances the GABA response at ␣2-, ␣3-, and ␣5-
receptors (McKernan et al., 2000). In line with a lack of
␣1-receptor activation, L-838,417 showed a high potency in
anxiolytic tests (elevated plus maze and fear-potentiated
startle) and in anticonvulsant tests (pentylenetetrazole, au-
diogenic seizures). However, even at doses that occupied 95%
of benzodiazepine sites, L-838,417 failed to impair motor
performance (rotarod test, chain-pulling test) (McKernan et
al., 2000). These findings are a major step forward. Ligands
with subtype-selective efficacy, which distinguish ␣2-, ␣3-,
and ␣5,-receptors from ␣1-receptors, provide a new way to
develop selective anxiolytics without a sedative component.
Further improvement may be achieved by focusing the ligand
affinity or efficacy more specifically on ␣2-receptors (see
above).
SL65.1498. The pyrido-indole-4-carboxamide derivative
SL65.1498 (6-fluoro-9-methyl-2phenyl-4-(pyrrolidin-1-yl-
carbonyl)-2,9-dihydro-1H-pyrido[3,4-b]indol-1-one) shows
Benzodiazepine Pharmacology 7
higher affinity for ␣1-, ␣2-, and ␣3-GABAA receptors com-
pared with ␣5-receptors. In addition, it acts as full agonist at
␣2- and ␣3-receptors but as a partial agonist at ␣1-GABAA
receptors. In line with its selectivity for the activation of ␣2-
and ␣3-receptors, the compound showed potent anxiolytic
action in animal models (punished lever pressing, punished
drinking, elevated plus maze, light/dark test) but did not
impair motor coordination (e.g., rotarod) or working memory
(Morris water maze) (Scatton et al., 2000).
Zaleplon. Zaleplon (CL 284,846) is a pyrazolopyrimidine
developed for the treatment of insomnia (Sanger et al., 1996).
At recombinant receptors, zaleplon binds preferentially to
␣1-receptors (␣1␤2␥2) and to receptors containing the ␥3-sub-
unit but binds 8- to 20-fold less to ␣2-, ␣3-, and ␣5-receptors
(Dämgen and Lüddens, 1999). Thus, zaleplon is largely a
ligand with preference for ␣1-receptors, which is in keeping
with its preponderant hypnotic activity. The contribution of
its interaction with ␥3-receptors is unclear since these recep-
tors are of low abundance in the brain.
Conclusions
The genetic dissection of the pharmacological functions of
GABAA receptor subtypes has opened up a new strategy in
drug development. The heuristic search for novel ligands at
the benzodiazepine site will be replaced by the rational tar-
geting of specific receptor subtypes. The ligand selectivity
can be achieved either by a preferential ligand affinity, pref-
erential ligand efficacy, or a mixture of both. Acting at small
subpopulations of GABAA receptors, these ligands are ex-
pected to lack the major side effects of the classical benzodi-
azepine drugs. The vision of specific anxiolytics with a re-
duced side-effect profile may become a reality. In addition,
therapeutic indications beyond those of the classical benzo-
diazepine drugs may emerge from subtype-specific drugs.
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Address correspondence to: Dr. Hanns Möhler, Institute of Pharmacology
and Toxicology, University of Zurich and Swiss Federal Institute of Technol-
ogy, Winterthurerstr. 190, 8057 Zurich, Switzerland. E-mail: mohler@pharma.
unizh.ch