Animal Models of Anxiety. In animal models of anxiety, most attention has focused on the ability of benzodiazepines to increase locomotor, feeding, or drinking behavior that has been suppressed by novel or aversive stimuli. In one paradigm, animal behaviors that previously had been rewarded by food or water are punished periodically by an electric shock. The time during which shocks are delivered is signaled by some auditory or visual cue, and untreated animals stop performing almost completely when the cue is perceived. The difference in behavioral responses during the punished and unpunished periods is eliminated by benzodiazepine receptor agonists, usually at doses that do not reduce the rate of unpunished responses or produce other signs of impaired motor function. Similarly, rats placed in an unfamiliar environment exhibit markedly reduced exploratory behavior (neophobia), whereas animals treated with benzodiazepines do not. Opioid analgesics and antipsychotic drugs do not increase suppressed behaviors, and phenobarbital and meprobamate usually do so only at doses that also reduce spontaneous or unpunished behaviors or produce ataxia.
The difference between the dose required to impair motor function and that necessary to increase punished behavior varies widely among the benzodiazepines and depends on the species and experimental protocol. Although such differences may have encouraged the marketing of some benzodiazepines as selective sedative-hypnotic agents, they have not predicted with any accuracy the magnitude of sedative effects among those benzodiazepines marketed as anxiolytic agents.
Tolerance to Benzodiazepines. Studies on tolerance in laboratory animals often are cited to support the belief that disinhibitory effects of benzodiazepines are distinct from their sedative-ataxic effects. For example, tolerance to the depressant effects on rewarded or neutral behavior occurs after several days of treatment with benzodiazepines; the disinhibitory effects of the drugs on punished behavior are augmented initially and decline after 3-4 weeks (File, 1985). Although most patients who ingest benzodiazepines chronically report that drowsiness wanes over a few days, tolerance to the impairment of some measures of psychomotor performance (e.g., visual tracking) usually is not observed. Whether tolerance develops to the anxiolytic effects of benzodiazepines remains a subject of debate. Many patients can maintain themselves on a fairly constant dose; increases or decreases in dosage appear to correspond with changes in problems or stresses. On the other hand, some patients either do not reduce their dosage when stress is relieved or steadily escalate dosage. Such behavior may be associated with the development of drug dependence (Chapter 24).
Some benzodiazepines induce muscle hypotonia without interfering with normal locomotion and can decrease rigidity in patients with cerebral palsy. In contrast to effects in animals, there is only a limited degree of selectivity in humans. Clonazepam in non-sedative doses does cause muscle relaxation, but diazepam and most other benzodiazepines do not. Tolerance occurs to the muscle relaxant and ataxic effects of these drugs.
Experimentally, benzodiazepines inhibit seizure activity induced by either pentylenetetrazol or picrotoxin, but strychnine- and maximal electroshock-induced seizures are suppressed only at doses that also severely impair locomotor activity. Clonazepam, nitrazepam, and nordazepam have more selective anticonvulsant activity than most other benzodiazepines. Benzodiazepines also suppress photic seizures in baboons and ethanol-withdrawal seizures in humans. However, the development of tolerance to the anticonvulsant effects has limited the usefulness of benzodiazepines in the treatment of recurrent seizure disorders in humans (Chapter 21).
Although analgesic effects of benzodiazepines have been observed in experimental animals, only transient analgesia is apparent in humans after intravenous administration. Such effects actually may involve the production of amnesia. Unlike barbiturates, benzodiazepines do not cause hyperalgesia.
Effects on the Electroencephalogram (EEG) and Sleep Stages. The effects of benzodiazepines on the waking EEG resemble those of other sedative-hypnotic drugs. Alpha activity is decreased, but there is an increase in low-voltage fast activity. Tolerance occurs to these effects.
Benzodiazepines decrease sleep latency, especially when first used, and diminish the number of awakenings and the time spent in stage 0 (a stage of wakefulness). Time in stage 1 (descending drowsiness) usually is decreased, and there is a prominent decrease in the time spent in slow-wave sleep (stages 3 and 4). Most benzodiazepines increase the time from onset of spindle sleep to the first burst of rapid-eye-movement (REM) sleep, and the time spent in REM sleep usually is shortened. However, the number of cycles of REM sleep usually is increased, mostly late in the sleep time. Zolpidem and zaleplon suppress REM sleep to a lesser extent than do benzodiazepines and thus may be superior to benzodiazepines for use as hypnotics (Dujardin et al., 1998).
Despite the shortening of stage 4 and REM sleep, benzodiazepine administration typically increases total sleep time, largely by increasing the time spent in stage 2 (which is the major fraction of non-REM sleep). The effect is greatest in subjects with the shortest baseline total sleep time. In addition, despite the increased number of REM cycles, the number of shifts to lighter sleep stages (1 and 0) and the amount of body movement are diminished. Nocturnal peaks in the secretion of growth hormone, prolactin, and luteinizing hormone are not affected. During chronic nocturnal use of benzodiazepines, the effects on the various stages of sleep usually decline within a few nights. When such use is discontinued, the pattern of drug-induced changes in sleep parameters may "rebound," and an increase in the amount and density of REM sleep may be especially prominent. If the dosage has not been excessive, patients usually will note only a shortening of sleep time rather than an exacerbation of insomnia.
Although some differences in the patterns of effects exerted by the various benzodiazepines have been noted, their use usually imparts a sense of deep or refreshing sleep. It is uncertain to which effect on sleep parameters this feeling can be attributed. As a result, variations in the pharmacokinetic properties of individual benzodiazepines appear to be much more important determinants of their effects on sleep than are any potential differences in their pharmacodynamic properties.
Molecular Targets for Benzodiazepine Actions in the CNS. Benzodiazepines appear to exert most of their effects by interacting with inhibitory neurotransmitter receptors directly activated by GABA (Chapter 14). The ionotropic GABAA receptors consist of five subunits that co-assemble to form an integral chloride channel (Figure 14–11). GABAA receptors are responsible for most inhibitory neurotransmission in the CNS. Benzodiazepines act at GABAA receptors by binding directly to a specific site that is distinct from that of GABA binding. Unlike barbiturates, benzodiazepines do not activate GABAA receptors directly; rather, benzodiazepines act allosterically by modulating the effects of GABA. Benzodiazepines and GABA analogs bind to their respective sites on brain membranes with nanomolar affinity. Benzodiazepines modulate GABA binding, and GABA alters benzodiazepine binding in an allosteric fashion.
Benzodiazepines and related compounds can act as agonists, antagonists, or inverse agonists at the benzodiazepine-binding site on GABAA receptors. Agonists at the binding site increase, and inverse agonists decrease, the amount of chloride current generated by GABAA-receptor activation. Agonists at the benzodiazepine binding site shift the GABA concentration-response curve to the left, whereas inverse agonists shift the curve to the right. Both these effects are blocked by antagonists at the benzodiazepine binding site. In the absence of an agonist or inverse agonist for the benzodiazepine binding site, an antagonist for this binding site does not affect GABAA receptor function. One such antagonist, flumazenil, is used clinically to reverse the effects of high doses of benzodiazepines. The behavioral and electrophysiological effects of benzodiazepines also can be reduced or prevented by prior treatment with antagonists at the GABA binding site (e.g., bicuculline).
The strongest evidence that benzodiazepines act directly on GABAA receptors comes from recombinant expression of cDNAs encoding subunits of the receptor complex, which resulted in high-affinity benzodiazepine binding sites and GABA-activated chloride conductances that were enhanced by benzodiazepine receptor agonists (Burt, 2003). The properties of the expressed receptors generally resemble those of GABAA receptors found in most CNS neurons. Each GABAA receptor is believed to consist of a pentamer of homologous subunits. Thus far 16 different subunits have been identified and classified into seven subunit families: six α, three β, three γ, and single δ, ∊, π, and θ subunits. Additional complexity arises from RNA splice variants of some of these subunits (e.g., γ2 and α6). The exact subunit structures of native GABA receptors still are unknown, but it is thought that most GABA receptors are composed of α, β, and γ subunits that co-assemble with some uncertain stoichiometry. The multiplicity of subunits generates heterogeneity in GABAA receptors and is responsible, at least in part, for the pharmacological diversity in benzodiazepine effects in behavioral, biochemical, and functional studies. Studies of cloned GABAA receptors have shown that the co-assembly of a γ subunit with α and β subunits confers benzodiazepine sensitivity to GABAA receptors (Burt, 2003). Receptors composed solely of α and β subunits produce functional GABAA receptors that also respond to barbiturates, but they neither bind nor are affected by benzodiazepines. Benzodiazepines are believed to bind at the interface between α and γ subunits, and both subunits determine the pharmacology of the benzodiazepine binding site (Burt, 2003). For example, receptors containing the α1 subunit are pharmacologically distinct from receptors containing α2, α3, or α5 subunits (Pritchett and Seeburg, 1990), reminiscent of the pharmacological heterogeneity detected with radioligand-binding studies using brain membranes. Receptors containing the α6 subunit do not display high-affinity binding of diazepam and appear to be selective for the benzodiazepine receptor inverse agonist RO 15-4513, which has been tested as an alcohol antagonist (Lüddens et al., 1990). The subtype of γ subunit also modulates benzodiazepine pharmacology, with lower-affinity binding observed in receptors containing the γ1 subunit. Although theoretically ~1 million different GABAA receptors could be assembled from all these different subunits, constraints for the assembly of these receptors apparently limit their numbers (Sieghart et al., 1999).
An understanding of which GABAA receptor subunits are responsible for particular effects of benzodiazepines in vivo has emerged. The mutation to arginine of a histidine residue at position 101 of the GABAA receptor α1 subunit renders receptors containing that subunit insensitive to the GABA-enhancing effects of diazepam (Kleingoor et al., 1993). Mice bearing these mutated subunits fail to exhibit the sedative, the amnestic, and in part the anticonvulsant effects of diazepam while retaining sensitivity to the anxiolytic, muscle-relaxant, and ethanol-enhancing effects. Conversely, mice bearing the equivalent mutation in the α2 subunit of the GABAA receptor are insensitive to the anxiolytic effects of diazepam (Burt, 2003). The attribution of specific behavioral effects of benzodiazepines to individual receptor subunits will aid in the development of new compounds exhibiting fewer undesired side effects.
Electrophysiological studies in vitro have shown that the enhancement of GABA-induced chloride currents by benzodiazepines results primarily from an increase in the frequency of bursts of chloride channel opening produced by submaximal amounts of GABA (Twyman et al., 1989). Inhibitory synaptic transmission measured after stimulation of afferent fibers is potentiated by benzodiazepines at therapeutically relevant concentrations. Prolongation of spontaneous miniature inhibitory postsynaptic currents (IPSCs) by benzodiazepines also has been observed. Although sedative barbiturates also enhance such chloride currents, they do so by prolonging the duration of individual channel-opening events. Macroscopic measurements of GABAA receptor-mediated currents indicate that benzodiazepines shift the GABA concentration-response curve to the left without increasing the maximum current evoked with GABA. These findings collectively are consistent with a model in which benzodiazepines exert their major actions by increasing the gain of inhibitory neurotransmission mediated by GABAA receptors.
GABAA receptor subunits also may play roles in the targeting of assembled receptors to their proper locations in synapses. In knockout mice lacking the γ2 subunit, GABAA receptors did not localize to synapses, although they were formed and translocated to the cell surface (Essrich et al., 1998). The synaptic clustering molecule gephyrin also plays a role in receptor localization.
GABAA Receptor-Mediated Electrical Events: in vivo Properties. The remarkable safety of the benzodiazepines is likely related to the fact that their effects in vivo depend on the presynaptic release of GABA; in the absence of GABA, benzodiazepines have no effects on GABAA receptor function. Although barbiturates also enhance the effects of GABA at low concentrations, they directly activate GABA receptors at higher concentrations, which can lead to profound CNS depression (discussed later). Further, the behavioral and sedative effects of benzodiazepines can be ascribed in part to potentiation of GABA-ergic pathways that serve to regulate the firing of neurons containing various monoamines (Chapter 14). These neurons are known to promote behavioral arousal and are important mediators of the inhibitory effects of fear and punishment on behavior. Finally, inhibitory effects on muscular hypertonia or the spread of seizure activity can be rationalized by potentiation of inhibitory GABA-ergic circuits at various levels of the neuraxis. In most studies conducted in vivo or in situ, the local or systemic administration of benzodiazepines reduces the spontaneous or evoked electrical activity of major (large) neurons in all regions of the brain and spinal cord. The activity of these neurons is regulated in part by small inhibitory interneurons (predominantly GABA-ergic) arranged in feedback and feedforward types of circuits. The magnitude of the effects produced by benzodiazepines varies widely depending on such factors as the types of inhibitory circuits that are operating, the sources and intensity of excitatory input, and the manner in which experimental manipulations are performed and assessed. For example, feedback circuits often involve powerful inhibitory synapses on the neuronal soma near the axon hillock, which are supplied predominantly by recurrent pathways. The synaptic or exogenous application of GABA to this region increases chloride conductance and can prevent neuronal discharge by shunting currents that otherwise would depolarize the membrane of the initial segment. Accordingly, benzodiazepines markedly prolong the period after brief activation of recurrent GABA-ergic pathways during which neither spontaneous nor applied excitatory stimuli can evoke neuronal discharge; this effect is reversed by the GABAA receptor antagonist bicuculline (see Figure 14–10).
The macromolecular complex containing GABA-regulated chloride channels also may be a site of action of general anesthetics, ethanol, inhaled drugs of abuse, and certain metabolites of endogenous steroids (Whiting, 2003). Among the latter, allopregnanolone (3α-hydroxy, 5α-dihydroprogesterone) is of particular interest. This compound, a metabolite of progesterone that can be formed in the brain from precursors in the circulation and also synthesized by glial cells, produces barbiturate-like effects, including promotion of GABA-induced chloride currents and enhanced binding of benzodiazepines and GABA-receptor agonists. As with the barbiturates, higher concentrations of the steroid activate chloride currents in the absence of GABA, and its effects do not require the presence of a γ subunit in GABAA receptors expressed in transfected cells. Unlike the barbiturates, however, the steroid cannot reduce excitatory responses to glutamate (discussed later). These effects are produced very rapidly and apparently are mediated by interactions at sites on the cell surface. A congener of allopregnanolone (alfaxalone) was used previously outside the U.S. for the induction of anesthesia.
Respiration. Hypnotic doses of benzodiazepines are without effect on respiration in normal subjects, but special care must be taken in the treatment of children (Kriel et al., 2000) and individuals with impaired hepatic function, such as alcoholics (Guglielminotti et al., 1999). At higher doses, such as those used for preanesthetic medication or for endoscopy, benzodiazepines slightly depress alveolar ventilation and cause respiratory acidosis as the result of a decrease in hypoxic rather than hypercapnic drive; these effects are exaggerated in patients with chronic obstructive pulmonary disease (COPD), and alveolar hypoxia and CO2 narcosis may result. These drugs can cause apnea during anesthesia or when given with opioids. Patients severely intoxicated with benzodiazepines only require respiratory assistance when they also have ingested another CNS-depressant drug, most commonly ethanol.
In contrast, hypnotic doses of benzodiazepines may worsen sleep-related breathing disorders by adversely affecting control of the upper airway muscles or by decreasing the ventilatory response to CO2. The latter effect may cause hypoventilation and hypoxemia in some patients with severe COPD, although benzodiazepines may improve sleep and sleep structure in some instances. In patients with obstructive sleep apnea (OSA), hypnotic doses of benzodiazepines may decrease muscle tone in the upper airway and exaggerate the impact of apneic episodes on alveolar hypoxia, pulmonary hypertension, and cardiac ventricular load. Many clinicians consider the presence of OSA to be a contraindication to the use of alcohol or any sedative-hypnotic agent, including a benzodiazepine; caution also should be exercised with patients who snore regularly, because partial airway obstruction may be converted to OSA under the influence of these drugs. In addition, benzodiazepines may promote the appearance of episodes of apnea during REM sleep (associated with decreases in oxygen saturation) in patients recovering from a myocardial infarction; however, no impact of these drugs on survival of patients with cardiac disease has been reported.
Cardiovascular System. The cardiovascular effects of benzodiazepines are minor in normal subjects except in severe intoxication; the adverse effects in patients with obstructive sleep disorders or cardiac disease were noted above. In preanesthetic doses, all benzodiazepines decrease blood pressure and increase heart rate. With midazolam, the effects appear to be secondary to a decrease in peripheral resistance, but with diazepam, they are secondary to a decrease in left ventricular work and cardiac output. Diazepam increases coronary flow, possibly by an action to increase interstitial concentrations of adenosine, and the accumulation of this cardiodepressant metabolite also may explain the negative inotropic effects of the drug. In large doses, midazolam decreases cerebral blood flow and oxygen assimilation considerably (Nugent et al., 1982).
GI Tract. Benzodiazepines are thought by some gastroenterologists to improve a variety of "anxiety related" gastrointestinal disorders. There is a paucity of evidence for direct actions. Benzodiazepines partially protect against stress ulcers in rats, and diazepam markedly decreases nocturnal gastric secretion in humans. Other agents are considerably more effective in acid-peptic disorders (Chapter 45).