GABAA Receptor Subtypes

GABAA receptors play a critical role in mediating inhibitory neurotransmission

and as targets of the anxiolytic benzodiazepines. The molecular structure of

GABAA receptors is shown in figure 9-21. Each subunit of a GABAA receptor has

four transmembrane regions (Figure 9-21A). When five subunits cluster together,

they form an intact GABAA receptor with a chloride channel in the center (Figure

9-21B). There are many different subtypes of GABAA receptors, depending upon

which subunits are present (Figure 9-21C). Subunits of GABAA receptors are

sometimes also called isoforms, and include a (with six isoforms, α1 to α6), β (with

three isoforms, β1 to β3) γ (with three isoforms, γ1 to γ3), δ, ε, π, θ, dan ρ (with

three isoforms, ρ1 to ρ3) (Figure 9-21C). What is important for this discussion is

that, depending upon which subunits are present, the functions of a GABAA

receptor can vary significantly.

Benzodiazepine-insensitive GABAA Receptors

Benzodiazepine-insensitive GABAA receptors are those with α4, α6, γ1, or δ subunits

(Figure 9-21C). GABAA receptors with a δ subunit rather than a γ subunit, plus

either α4 or α6 subunits, do not bind to benzodiazepines. Such GABAA receptors do

bind to other modulators, namely the naturally occurring neurosteroids, as well as

to alcohol and to some general anesthetics (Figure 9-21C). The binding site for

these non-benzodiazepine modulators is located between the α and the δ subunits,

one site per receptor complex (Figure 9-21C). Two molecules of GABA bind per

receptor complex, at sites located between the α and the β subunits, sometimes

referred to as the GABA agonist site (Figure 9-21C). Since the site for the

modulators is in a different location from the agonist sites for GABA, the

modulatory site is often called allosteric (literally, “other site”), and the agents

that bind there are called allosteric modulators.

Benzodiazepine-insensitive GABAA receptor subtypes (with δ subunits)

are located extrasynaptically, where they capture not only GABA that diffuses

away from the synapse, but also neurosteroids synthesized and released by glia

(Figure 9-22). Extrasynaptic, benzodiazepine-insensitive GABAA receptors are

thought to mediate a type of inhibition at the postsynaptic neuron that is tonic, in

contrast to the phasic type of inhibition mediated by postsynaptic benzodiazepine-

sensitive GABAA receptors (Figure 9-22). Thus, tonic inhibition may be regulated

by the ambient levels of extracellular GABA molecules that have escaped

presynaptic reuptake and enzymatic destruction. Tonic inhibition is thought to set

the overall tone and excitability of the postsynaptic neuron, and to be important

for certain regulatory events such as the frequency of neuronal discharge in

response to excitatory inputs.

Since the GABAA receptors that modulate this action are not sensitive

to benzodiazepines, they are not likely to be involved in the anxiolytic actions of

benzodiazepines in various anxiety disorders. However, novel hypnotics as well

as anesthetics have targeted these extrasynaptic benzodiazepine-insensitive

GABAA receptors, and it is possible that novel synthetic neurosteroids that also

target benzodiazepine-insensitive GABAA receptor subtypes could some day

become novel anxiolytics. Indeed, anxiety itself may in part be dependent upon

having the right amount of tonic inhibition in key anatomic areas such as the

amygdala and cortical areas of CSTC loops. Furthermore, naturally occurring

neurosteroids may be important in setting that inhibitory tone in critical brain

areas. If this tone becomes dysregulated, it is possible that abnormal neuronal

excitability could become a factor in the development of various anxiety

disorders.

Benzodiazepine-sensitive GABAA Receptors

Benzodiazepine-sensitive GABAA receptors have several structural and

functional features that make them distinct from benzodiazepine-insensitive

GABAA receptors. In contrast to benzodiazepine-insensitive GABAA

receptors, for a GABAA receptor to be sensitive to benzodiazepines, and thus to

be a target for benzodiazepine anxiolytics, there must be two β units plus a γ

unit of either the γ2 or γ3 subtype, plus two α units of the α1, α2, or α3,

subtype (Figure 9-21C). Benzodiazepines appear to bind to the region of

the receptor between the γ2/3 subunit and the α1/2/3 subunit, one benzodiazepine

molecule per receptor complex (Figure 9-21C). GABA itself binds with two

molecules of GABA per receptor complex to the GABA agonist sites in the

regions of the receptor between the α and the β units (Figure 9-21C).

Benzodiazepine-sensitive GABAA receptor subtypes (with γ subunits and α I-3

subunits) are thought to be postsynaptic in location and to mediate a type of

inhibition at the postsynaptic neuron that is phasic, occurring in bursts of

inhibition that are triggered by peak concentrations of synaptically released

GABA (Figure 9-22). Theoretically, benzodiazepines acting at these receptors,

particularly the α2/3 subtypes clustered postsynaptic GABA sites, should exert

an anxiolytic effect due to enhancement of phasic postsynaptic inhibition. If

this action occurs at overly active output neurons in the amygdala or in

CSTC loops, it would theoretically cause anxiolytic actions with reduction of

both fear and worry.

Not all benzodiazepine-sensitive GABAA receptors are the same. Notably, those

benzodiazepine-sensitive GABAA receptors with α l subunits may be most

important for regulating sleep and are the presumed targets of numerous

sedative-hypnotic agents, including both benzodiazepine and non-

benzodiazep positive allosteric modulators of the GABAA, receptor (Figure 9-

21C). The α1 subtype of GABAA receptors and the drugs that bind to it are

discussed further in Chapter 11 on sleep. Some of these agents are selective for

only the α1 subtype of GABAA receptor.

On the other hand, benzodiazepine-sensitive GABAA receptors with α2

(and/or α3) subunits may be most important for regulating anxiety and are the

presumed targets of the anxiolytic benzodiazepines (Figure 9-21C). However,

currently available benzodiazepines are nonselective for GABAA receptors with

different α subunits. Thus, there is an ongoing search for selective α2/3 agents that

could be utilized to treat anxiety disorders in humans. Such agents would theor-

etically be anxiolytic without being sedating. Partial agonists selective for α2/3

subunits of benzodiazepine-sensitive GABAA receptors hypothetically would

cause less euphoria, be less reinforcing and thus less abusable, cause less

dependence, and cause fewer problems in withdrawal. Such agents are being

investigated but have not yet been introduced into clinical practice. Abnormally

expressed γ2, α2, or δ subunits have all been associated with different types of

epilepsy. Receptor subtype expression can change in response to chronic

benzodiazepine administration and withdrawal, and could theoretically be

altered in patients with various anxiety-disorder subtypes.

Benzodiazepines as positive allosteric modulators or PAMs

Since the benzodiazepine-sensitive GABAA receptor complex is regulated not

only by GABA itself, but also by benzodiazepines at a highly specific allosteric

modulator binding site (Figure 9-23), this has led to the notion that there may

be an "endogenous" or naturally occurring benzodiazepine synthesized in the

brain (the brain's own Xanax!). However, the identity of any such substance

remains elusive. Furthermore, it is now known that synthetic drugs that do not

have a benzodiazepine structure also bind to the "benzodiazepine receptor."

These developments have led to endless confusion with terminology, since non-

benzodiazepines also bind to the "benzodiazepine receptor!" Thus, many experts

now call the "benzodiazepine site" the GABAA allosteric modulatory site and

anything that binds to this site, including benzodiazepines, allosteric modulators.

Acting alone, GABA can increase the frequency of opening of the chloride

channel, but only to a limited extent (compare Figure 9-23A and B). The

combination of GABA with benzodiazepines is thought to increase the frequency

of opening of inhibitory chloride channels but not to increase the conductance

of chloride across individual chloride channels, nor to increase the duration

of channel opening. The end result is more inhibition. More inhibition

supposedly yields more anxiolytic action. How does this happen? The answer

is that benzodiazepines act as agonists at the allosteric modulatory site of

GABA binding. They are positive allosteric modulators, or PAMs, but have no

activity on their own. Thus, when benzodiazepines bind to the allosteric

modulatory site, they have no activity when GABA is not simultaneously

binding to its agonist sites (compare Figare 9-23A and C).

So, how do benzodiazepines act as PAMs? This can occur only when GABA

is binding to its agonist sites. The combination of benzodiazepines at the

allosteric site plus GABA at its agonist sites increases the frequency of opening

of the chloride channel to an extent not possible with GABA alone (compare

Figure 9-23B and D).

The actions of benzodiazepines essentially as agonists at their positive

allosteric sites can be reversed by the neutral antagonist flumazenil (Figure 9-

24). Flumazenil is a short-acting intravenously adminis tered antagonist to

benzodiazepines that can reverse overdoses or anesthesia from benzodiazepines

but can also induce seizures or withdrawal in patients dependent upon

benzodiazepines.

Benzodiazepines as anxiolytics

A simplified notion of how benzodiazepine anxiolytics might modulate

excessive output from the amygdala during fear responses in anxiety disorders

is shown in Figure 9-25. Excessive amygdala activity (shown in Figures 9-8

through 9-12 and in Figure 9-25A) is theoretically reduced by enhancing the

phasic inhibitory actions of benzodiazepine PAMs at postsynaptic GABAA

receptors within the amygdala to blunt fear-associated outputs, hypothetically

reducing the symptom of fear (Figure 9-25B). Benzodiazepines also theoretically

modulate excessive output from worry loops (Figure 9-26A) by enhancing the

actions of inhibitory interneurons in CSTC circuits (Figure 9-26B), hypothetically

reducing the symptom of worry.

Alpha-2-delta figands as anxiolytics

We have discussed voltage-sensitive calcium channel (VSCCs) in Chapter

3 and have illustrated presynaptic N and P/Q subtypes of VSCC and their

role in excitatory neurotransmitter release (see Figures 3-19 and 3-22

through 3-24). Gabapentin and pregabalin, also known as α2δ ligands, since

they bind to the α2δ subunit of presynaptic N and P/Q VSCCs, block the

release of excitatory neurotransmitters such as glutamate when

neurotransmission is excessive, as postulated in the amygdala to cause fear

(Figure 9-25A) and in CSTC circuits to cause worry (Figure 9-26A).

Hypothetically, α2δ ligands bind to open, overly active VSCCs in the

amygdala (Figure 9-25C) to reduce fear, and in CSTC circuits (Figure 9-26C) to

reduce worry. The α2δ ligands pregabalin and gabapentin have demonstrated

anxiolytic actions in social anxiety disorder and panic disorder, and are

also proven to be effective for the treatment of epilepsy and certain pain

conditions, including neuropathic pain and fibromyalgia. The actions of α2δ

ligands on VSCCs are discussed in Chapter 10 on pain and illustrated in

Figures 10-17 through 10-19. α2δ ligands clearly have different

mechanisms of action compared to serotonin reuptake inhibitors or

benzodiazepines, and thus can be useful for patients who do not do well on

SSRIs/SNRIs or benzodiazepines. Also, α2δ ligands can be useful to combine with

SSRIs/SNRIs or benzodiazepines in patients who are partial responders and

are not in remission.

Serotonin and anxiety

Since the symptoms, circuits, and neurotransmitters Liked to anxiety disorders

overlap extensively with those for major depressive disorder (Figure 9-1), it is

not surprising that drugs developed as antidepressants have proven to be

effective treatments for anxiety disorders. Indeed, the leading treatments for

anxiety disorders today are increasingly drugs originally developed as

antidepressants. Serotonin is a key neurotransmitter that innervates the

amygdala as well as all the elements of CSTC circuits, lamely, prefrontal cortex,

striatum, and thalamus, and thus is poised to regulate both fear and worry

serotonin pathways are discussed in Chapters 5 and 6 and illustrated in

Figure 6-33). Antidepressants that can increase serotonin output by locking

the serotonin transporter (SERT) are also effective in reducing symptoms of

anxiety and fear n every one of the anxiety disorders illustrated in Figures 9-2

though 9-5 - namely, GAD, panic disorder, social anxiety disorder, and PTSD.

Such agents include the well-known SSRIs (selective serotonin reuptake

inhibitors; discussed in Chapter 7 and their mechanism of action illustrated in

Figures 7-12 through 7-17), as well as the SNRIs (serotonin-norepinephrine

reuptake inhibitors; also discussed in Chapter 7 and their mechanism of

action illustrated in Figures 7-12 through 7-17 plus Figures 7-33 and 7-34).

A serotonin IA (5HT IA) partial agonist, buspirone, is recognized as a

generalized anxiolytic, but not as a treatment for anxiety disorder subtypes.

5HT1A partial agonists as augmenting agents to antidepressants are discussed in

Chapter 7, as are antidepressants combining 5HTIA partial agonism with serotonin

reuptake inhibition (i.e., SPARIs and vilazodone: see Figures 7-25 through 7-29),

which should theoretically be anxiolytic as well as antidepressant in action. The

5HTIA partial agonist properties of numerous atypical antipsychotics are discussed

in Chapter 5 and illustrated in Figures 5-15, 5-16, 5-25, and 5-26.

The potential anxiolytic actions of buspirone could theoretically be due to

5HTIA partial agonist actions at both presynaptic and postsynaptic 5HTIA receptors

(Figure 9-27 and Figures 5-15, 5-16, 5-25, 7-25 through 7-29), with actions at

both sites resulting in enhanced serotonergic activity in projections to the

amygdala (Figure 9-25D), prefrontal cortex, striatum, and thalamus (Figure 9-

26D). SSRIS and SNRIs theoretically do the same thing (Figures 9-25D and

9-26D). Since the onset of anxiolytic action for buspirone is delayed,

just as it is for antidepressants, this has led to the belief that 5HT 15

agonists exert their therapeutic effects by virtue of adaptive neuronal

events and receptor events (Figures 7-12 through 7-17 and 7-25 through 7-

29), rather than simply by the acute occupancy of 5HT1A receptors. In this

way, the presumed mechanism of action of 5HT IA partial agonists is analo-

gous to the antidepressants - which are also presumed to act by

adaptations in neurotransmitter receptors - and different from the

benzodiazepine anxiolytics - which act relatively acutely by occupying

benzodiazepine receptors.

Noradrenergic hyperactivity in anxiety

Norepinephrine is another neurotransmitter with important regulatory

input to the amygdala (Figure 9-28) and to the prefrontal cortex and

thalamus in CSTC circuits (Figure 9-29). Excessive noradrenergic output from

the locus coeruleus can not only result in numerous peripheral

manifestations of autonomic overdrive, as discussed above and illustrated

in Figures 9-8 through 9-12 but can also trigger numerous central symptoms

of anxiety and fear, such as nightmares. hvperarousal states, flashbacks,

and panic attacks (Figure 9-28A). Excessive noradrenergic activity can also

reduce the efficiency of information processing in the pre frontal cortex and

thus in CSTC circuits and theorettically cause worry (Figure 9-29A).

Hypothetically, these symptoms may be mediated in part by excessive

noradrenergic input onto α1 and β1 adrenergic Postsynaptic receptors in the

amygdala (Figure 9-28A) or prefrontal cortex (Figure 9-29A). Symptoms of

hyperarousal such as nightmares can be reduced in some patients with α1

adrenergic blockers such as Prazocin (Figure 9-28B); symptoms of fear

(Figure 9,28C) and worry (Figure 9-2913) can be reduced by norepinephrine

reuptake inhibitors (also called NET or norepinephrine transporter

inhibitors). The clinical effects of NET inhibitors can be confusing,

because symptoms of anxiety can be made transiently worse immediately

following initiation of an SNRI or selective NET inhibitor, when

noradrenergic activity is initially increased but the post-synaptic receptors

have not yet adapted. However, these same NET inhibitory actions, if

sustained over time, will down regulate and desensitize postsynaptic NE

receptors such as β1, receptors, and actually, reduce symptoms of fear and

worry long term (Figure 9-29B).

Fear conditioning versus fear extinction

Fear conditioning

Fear conditioning is a concept as old as Pavlov's dogs. If an aversive stimulus such

as foot shock is coupled with a neutral stimulus such as a bell, the animal learns to

associate the two and will develop fear when it hears a bell. In humans, fear is

learned during stressful experiences associated with emotional trauma and is

influenced by an individual’s genetic predisposition as well as by an individual’s

prior exposure to environmental stressors that can cause stress sensitization of

brain circuits (e.g., child abuse: see Chapter 6 and Figures 6-40 through 6-43).

Often, fearful situations are managed successfully and then forgotten. Some fears

are crucial for survival, such as appropriately fearing dangerous situations, and

thus the mechanism of learned fear, called fear conditioning, has been

extremely well conserved across species, including humans. However, other

fears that are “learned” and not “forgotten” may hypothetically progress to

anxiety disorders or a major depressive episode. This is a big problem, since

almost 30% of the population will develop an anxiety disorder, due in large

part to stressful environments, including exposure to fearful events during

normal activities, in twenty-first-century society, but in particular during

war and natural disasters. Hearing an explosion, smelling burning rubber,

seeing a picture of a Wounded civilian, and seeing or hearing flood waters are

all sensory experiences than can trigger traumatic re-experiencing and

generalized hyperarousal and fear in PTSD. Panic associated with social

situations Will “teach” the patient to panic in social situations in social

anxiety disorder. Panic randomly associated With an attack that happens to

occur in a crowd, on a bridge, or in a shopping center will also trigger

another panic attack when the same environment is encountered in panic

disorder. These and other symptoms of anxiety disorders are all forms of

learning known as fear conditioning (Figure 9-30).

The amygdala is involved in “remembering” the various stimuli

associated with a given fearful situation. It does this by increasing the

efficiency of neurotransmission at glutamatergic synapses in the lateral

amygdala as sensory input about those stimuli comes in from the thalamus

or sensory cortex (Figure 9-30). This input is then relayed to the central amygdala,

where fear conditioning also improves the efficiency of neurotransmission at

another glutamate synapse there (Figure 9-30). Both synapses are

restructured and permanent learning is embedded into this circuit by

NMDA receptors triggering long term potentiation and synaptic plasticity, so

that subsequent input from the sensory cortex and thalamus is very

efficiently processed to trigger the fear response as output from the

central amygdala every time there is sensory input associated with the ori -

ginal fearful event (Figure 9-30; see also Figures 9-8 through 9-13).

Input to the lateral amygdala is modulated by the prefrontal cortex, especially

the ventromedial prefrontal cortex (VMPFC), and by the hippocampus. If the

VMPFC is unable to suppress the fear response at the level of the amygdala,

fear conditioning proceeds. The hippocampus remembers the context of the fear

conditioning and makes sure fear is triggered when the fearful stimulus and all

its associated stimuli are, encountered. Most contemporary

psychopharmacological treatments for anxiety and fear act by suppressing the fear

output from the amygdale (figures 9-25 and 9-28) and therefore are not cures, sice

the fundamental neuronal learning underlying fear conditioning in these patient

remains in place.