Discuss the role of the GABAA receptor chloride channel complex in the action of anxiolytic drugs

University Degree Subjects allied to Medicine

Introduction

Anxiolytic drugs are among the most frequently prescribed substances, used regularly by upwards of 10% of the population in most developed countries. This may be a consequence of the difficulties in classifying the difference between a pathological state of anxiety and a normal state of anxiety.

The core manifestation of anxiety is experience of excessive anxiety, with other symptoms including: restlessness and agitation; tachycardia; sweating; gastrointestinal disorders; sleep disturbances; and the interference of normal productive activities of the patients every day life.

Clinical conditions related to anxiety include phobic anxiety where anxiety is triggered by specific circumstances such as open spaces or spiders, and panic disorder which are attacks of overwhelming fear which occur in association with marked somatic symptoms such as sweating, tachycardia, chest pains, trembling, choking etc. The distinction between these conditions and generalised anxiety disorders is not well defined, and anxiolytic drugs are used to treat all of them.

Animal Models

Anxiety is a subjective human phenomenon and except for some of the associated somatic and autonomic changes, it has no obvious counterpart in experimental animals. In biological terms, anxiety may be defined as a form of behavioural inhibition that occurs in response to situations that are “new, non-rewarding or punishing”. In animal models this behavioural inhibition translates as immobility, or suppression of a behavioural response such as bar-pressing to obtain food. In order to develop new anxiolytic drugs, it is important animal tests are available that give a good guide to activity in man. Examples of animal tests used to demonstrate anxiety are the rotarod and grip strength test, activity cages to measure locomotor activity, the elevated plus maze test, the fear potentiated startle paradigm, testing the righting reflex of mice, and the light-dark choice test where “anxious” mice appear to choose to stay in dark areas rather than light ones.

Neuroanatomy of anxiety

The functional anatomy involves amydala-based neurocircuits with critical reciprocal connections to the medial prefrontal cortex.

Sensory information from the sensory thalamus and cortical areas converge on the lateral nucleus of the amygdala (LeDoux 1998). This includes information from all sensory modalities (except olfactory, which projects to periamygdaloid cortex). The lateral nucleus projects to the basal, accessory basal, and central nuclei. The central nuclei coordinates information from multiple nuclei to generate a behavioural response to the stimuli, using projections to the lateral hypothalamus (enhanced autonomic activity); periaqueductal grey (freezing and other motoric responses); and paraventricular nucleus (stress-related CRF release).

This type of networked circuitry allows neurochemical modulators to affect activity within each brain area and at points along the entire system.

GABA

Gamma-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the mammalian CNS. GABA participates in the regulation of neuronal excitability through interaction with specific membrane proteins (the GABAA receptors). The binding of GABA to these postsynaptic receptors results in an opening of a chloride channel integrated in the receptor which allows the entry of Cl- and consequently leads to hyperpolarization of the recipient cell. The action of GABA is allosterically modulated by a wide variety of chemical entities which interact with distinct binding sites at the GABAA receptor complex.

Several drugs such as benzodiazepines, barbiturates, neurosteroids, ethanol, some ...

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GABA

Several drugs such as benzodiazepines, barbiturates, neurosteroids, ethanol, some of the anticonvulsants, and general anesthetics interact with GABA receptors so as to elicit their pharmacological effects.

GABAA receptors

GABAA receptors are ubiquitous in the CNS, and so a major goal in neuropharmacology (and specifically in the treatment of anxiety) has been to target drugs selectively to define GABAA receptor subtypes and thereby refine the therapeutic spectrum of the presently available drugs, reduce the side effects and discover new therapeutic indications. GABAA receptors are pentameric membrane proteins, most clearly distinguished by their subunit architecture, which in mammalian brain comprises seven different classes of subunits with mostly multiple variants (α1-6, β1-3, γ1-3, ρ1-3, δ, ε, θ). The pentameric GABAA receptor assembly can be derived from a permutation and combination of two, three, four, or even five different subunits. Composition of various GABAA receptor assemblies can differ not only in different parts of the brain or in different cells but also in the same cell. Most GABAA receptors are made up of α-, β- and γ-subunits. Various techniques such as mutation, gene knockout and inhibition of GABAA receptor subunits by antisense oligodeoxynucleotides have been used to establish the physiological/pharmacological significance of the GABAA receptor subunits and their native receptor assemblies in vivo. Radio-ligand binding to the immunoprecipitated receptors, co-localization studies using immunoaffinity chromatography, and immunocytochemistry techniques have been utilized to establish the composition and pharmacology of native GABAA receptor assemblies.

Benzodiazepines

Benzodiazepines are the most commonly used group of anxiolytic and hypnotic agents. The first benzodiazepine, chlordiazepoxide, was synthesised by accident in 1961, the unusual 7-membered ring having been produced as a result of an unplanned reaction in the laboratories of Hoffman la Roche. Benzodiazepines have been found to cause a reduction of anxiety and aggression, sedation leading to improvement of insomnia, muscle relaxation and loss of motor coordination and suppression of convulsions. They act by binding to a specific regulatory site on the GABAA receptor, thus enhancing the inhibitory effect of GABA. Compared to other benzodiazepines, they are relatively safe in overdose, but their main disadvantages are interaction with alcohol which can enhance the depressant effect of alcohol in a more than additive way and cause life threatening respiratory depression, long-lasting hangover effects and the development of dependence and tolerance. Other side-effects include drowsiness, confusion, amnesia and impaired coordination which considerably affects manual skills such as driving performance.

One of the most thoroughly investigated modulatory site on the GABAA receptor complex is the benzodiazepine binding site.

Neurons that express exclusively α3-contianing receptors are located in the reticular activating system (i.e. noradrenergic, serotonergic, dopaminergic neurons). Previously it had been suggested that the anxiolytic effect of diazepam is due to the dampening in particular of the noradrenergic neurons in the locus coeruleus and its interactions with serotonergic neurons (Robbins et al 1995). However, in α3(H126R) mice the anxiolytic activity of diazepam, as tested by the light-dark choice test and the elevated plus-maze test, was not impaired compared with wild-type mice

Consequently, there has been an intensive search for modulatory agents with an improved profile, and a diversity of chemical entities distinct from the benzodiazepines, but with GABA modulatory effects have been identified. The existence of endogenous ligands for the GABAA receptor complex beside GABA has often been described, but their role in the regulation of GABA action is still a matter of controversy. The progress of molecular biology during the last decade has contributed enormously to the understanding of benzodiazepine receptor pharmacology. A total of 14 GABAA receptor subunits have been cloned from mammalian brain and have been expressed/co-expressed in stable cell lines. These transfected cells constitute an important tool in the characterization of subtype selective ligands. In spite of the rapidly expanding knowledge of the molecular and pharmacological mechanisms involved in GABA/benzodiazepine related CNS disorders, the identification of clinically selective acting drugs is still to come.

Neurosteroids

Neuroactive steroids are a novel class of positive allosteric modulators of the GABAA receptor. Although neuroactive steroids are endogenous neuronal modulators, synthetic entities with improved oral bioavailability have recently been developed. These compounds demonstrate efficacy as anticonvulsants against a range of convulsant stimuli and demonstrate anti-epileptogenic activity in a kindling model of epilepsy. Efficacy has also been reported in preclinical models of anxiety, insomnia, migraine and drug dependence

Genetic Models

In gene knock out experiments, removal of a particular receptor subunit would be expected to perturb the structure of a defined group of GABAA receptors and produce a corresponding alteration in the physiology and pharmacology of the mutant mice. Mice deficient on both the γ2s- and γ2l-subunits are entirely devoid of a response to benzodiazepines as shown behaviourally and in cultured dorsal root ganglion cells. This response highlights the need of the γ2-subunit for the formation of the benzodiazepine site of the GABAA receptors.

Although studies with knockout mice can provide important information on GABAA receptor mediated functions, some limitations exist. The knockout mutation might trigger adaptive changes during development and in neuronal function, and such a complex phenotype makes it difficult to draw unbiased conclusions about the function of an individual GABAA receptor subtype. For β3 and γ2 knockout mice, the phenotype is largely lethal and the behaviour of the few surviving animals might not necessarily be representative of the mutation but could instead reflect an accidental constellation of genetic and possibly environmental factors. Also, all GABAA receptor knockouts explored so far have a “neomycin resistance cassette” in the mutated gene. The presence of this cassette (a positive selection marker in embryonic stem cells) might in some cases alter the expression of neighbouring genes possibly via its own regulatory elements. Due to the fact that genes encoding GABAA receptor subunits are clustered at the chromosomal level, the expression of neighbouring genes encoding the other GABAA receptor subunits might be affected by the neomycin resistance cassette in the knockout mice. This is most probably the case for the gene encoding the α6- subunit, which is part of the α1, α6, β2, γ2 gene cluster. Therefore, although the knockout mice can provide interesting information on receptor assembly and function and on compensatory adaptations, these adaptations might largely preclude a molecular interpretation of drug responses with regard to a particular receptor.

Knock-in mutations are the replacement of a single amino acid codon in a defined gene in vivo. Studies on recombinant GABAA receptors have indicated that a His to Arg point mutation in the benzodiazepine binding site of GABAA receptors abolished binding of classical benzodiazepines but did not appear to affect receptor assembly and sensitivity to GABA. As a result the corresponding knock-in point mutation is not expected to be susceptible to appreciable changes in brain development or function. Knock-in point mutations have therefore been chosen as a popular strategy to dissect the pharmacology of GABAA receptor subtypes. The majority of GABAA receptors contain a binding site for diazepam and other related classical benzodiazepines that is located at the interface of the γ2-subunit and the respective α-subunit. These α-subunits contain a common feature, a conserved histidine residue in the drug-binding domain. Its conversion to an arginine residue renders the respective receptor diazepam insensitive in vitro. Exploiting this molecular switch, the His to Arg point mutation was introduced into the germ line of mice in the genes that encode the α1, 2, and 3 subunits. These mouse lines were expected to lack benzodiazepine effects that are normally mediated by the receptor subtype that contains the respective α-subunit. The receptors that contained the mutated subunits displayed a distribution of diazepam insensitive binding sites corresponding to that of wild type receptors as shown for α1, 2 and 3 containing point mutated receptors. Most importantly, the physiology of the neuronal circuits appeared to be unaffected in the point-mutated mouse lines. The GABA-induced responses of the mutated receptors were unaltered in cells expressing the α1 or the α2 containing receptor (Low et al 2000), which indicates that the operation of the GABA-gated ion channels by the physiological ligand has remained unchanged. The affinity for diazepam was reduced by a factor of at least 300, however it is currently not known how the presence of a point mutated α-subunit would affect the regulation of GABA induced Cl- currents by benzodiazepines.

Future development…

New insights into the sub-type specificity of benzodiazepine actions will provide precise guidelines for the development of new drugs with more selective actions and fewer side effects than those used currently in clinical practise. An important factor in anxiolytic profiling is the avoidance of a response at α1-containing receptors in favour of α2-, α3-, and α5-containing receptors. McKernan et al in 2000 developed and characterised the new ligand L838417, which is a break though in this direction. L838417 binds with high affinity to α1, α2, α3 and α5 containing receptors, but not to α4 or α6 containing receptors, and it is similar in this respect to diazepam.

However, in contrast to diazepam which is a full agonist at all benzodiazepine-sensitive GABAA receptors, L838417 fails to enhance the GABA response at α1 containing receptors but acts on α2, 3, and 5 containing receptors apparently with partial agonistic activity. L838417 displayed anxiolytic-like activity in wild type rats as shown in the elevated plus-maze test and in conditioned fear potentiated startle protocol. The behavioural characterisation of L838417 supports the conclusion that the sedative but not the anxiolytic-like properties of benzodiazepines are mediated by α1 containing GABAA receptors (McKernan et al 2000). It can be expected that more advanced anxiolytic drugs would be agonists selective for α2 containing GABAA receptors.

Also, sub-type specific drugs should display fewer side-effects, such as tolerance and dependence liability and depression of respiration, due to the fact that they only act on a small population of GABAA receptors.

In the future it may be possible for subtype specific ligands to be used in the treatment of not only anxiety and depression which are traditionally treated with benzodiazepines, but other neuropsychiatric disorders for which treatment has been relatively unsuccessful to date.