The neuroanatomical basis of anxiety

Pharmac. Ther.Vol. 55, pp. 149-181,1992 Printed in Great Britain. All rights reserved

0163-7258/92$15.00 © 1993PergamonPressLtd

Associate Editor: D. J. K. BALFOUR

THE N E U R O A N A T O M I C A L BASIS OF A N X I E T Y JUDITH A. PRATT

Department of Physiology and Pharmacology, University of Strathclyde, Glasgow, G1 IXW, U.K. Abstract--This review details the neural systems that are important in anxiety-related behaviours. In particular, the role of the amygdaloid complex, Papez circuit, septohippocampal formation and raphe nuclei are described and discussed. Evidence is gathered from a variety of experimental approaches. These include behavioural assessment of anxiety in animals after intracerebral injection of pharmacological agents and following lesions of discrete brain nuclei and selective neurotransmitter pathways. Further evidence is provided by functional brain mapping studies applied to animals and humans. It is proposed that the neural systems recruited in different experimental conditions of anxiety may differ, supporting the notion that clinical anxiety exists in several forms. This has implications for the identification of new anxiolytic treatments. In particular, the findings suggest that approaches aimed at identifying new anxiolytic agents must take into account both the distribution of receptors for the drug and the neuronal systems activated by the experimental protocol.

CONTENTS 1. Introduction 1.1. Anxiety: Forms and components 1.2. The use of animal behavioural models to assess anxiety 2. Neural Pathways Involved in Anxiety-related Behaviours: Evidence from Electrical Stimulation and Lesion Studies 2.1. Historical perspective 2.2. Components of the Papez circuit 2.3. Amygdala 2.3.1. Introduction 2.3.2. Amygdala and learned behaviours 2.3.3. The amygdala as part of the circuitry involved in conditioned fear 2.3.4. The amygdala, fear potentiated startle and other models of anxiety 2.4. Periaqueductal gray 2.5. Septohippocampal system 2.6. Raphe nuclei 2.7. Locus coeruleus 2.7.1. Electrical stimulation and lesion studies 2.7.2. Responses of noradrenergic neurons to stressful stimuli 2.7.3. Noradrenergic function in patients with anxiety disorders 3. Pharmacological Manipulation of 5-HT Systems and Anxiety 3.1. 5-HT antagonists and reduction of 5-HT function 3.2. Enhancement of 5-HT function 3.3. 5-HT receptor subtype-specific ligands 3.4. Interaction between benzodiazepines and 5-HT systems 4. Neuroanatomical Sites of Action of Anxiolytic and Anxiogenic Drugs in Modifying Anxiety-related Behaviours: Focal Injection Studies 4.1. Benzodiazepine-receptor ligands 4.1.1. Neuroanatomical locations of receptors

150 150 151 152 152 153 154 154 155 155 156 158 159 160 162 162 163 163 164 164 164 164 165 167 167 167

Abbreviations--CBF, cerebral blood flow; DHT, dihydroxytryptamine; FG 7142, N-methyl-fl-carboline-3carboxamide; GABA, 7-aminobutyric acid; 8-OH-DPAT, 8-hydroxy-2-(di-n-propylamino)tetralin; 5-HT, 5-hydroxytryptamine; 5-MeODMT, 5-methoxy,N,N-dimethyltryptamine; PAG, periaqueductal grey; PCPA, parachlorophenylalanine; PET, positron emission tomography. 149

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J.A. PRATT 4.1.2. Focal intracerebral injections of benzodiazepines 4.2. 5-HT-receptor ligands 5. Functional Brain Mapping During Anxiety and Related Behaviours 5.1. The influence of anxiogenic drugs and stressful stimuli upon local rates of cerebral functional activity in rodents 5.2. Positron emission tomography applied to investigate the neuroanatomical basis of anxiety and panic in humans 6. Summary and Conclusions Acknowledgements References

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1. INTRODUCTION Since the proposal by Papez in 1937 of the importance of the circuit involving the hypothalamus, anterior thalamus, cingulate gyrus and hippocampus in the experience and expression of emotion, the role of the limbic system has been the subject of many investigations aimed at elucidating the neural basis of anxiety. Researchers have, however, been limited by a number of problems. Firstly, the limitations in our ability to investigate neuronal function in the living human brain. Secondly, the difficulties in extrapolating findings from animal studies to those of the clinical situation and thirdly the evolving definition of what we understand by the term. Nevertheless, significant progress has been made in recent years of our understanding of the neuroanatomical pathways that underlie anxiety. In this review I have focussed on evidence from three different experimental approaches. Firstly, evidence derived from the ability of lesions and electrical stimulation of discrete brain structures and pathways to modify anxiety-related behaviours. Secondly, the ability of focal intracerebral injections of anxiolytic drugs to modify anxiety. Finally, the contribution that functional brain mapping studies in both animals and humans have made to our understanding of the neuroanatomical basis of anxiety-disorders is discussed. Prior to discussing these findings it is pertinent to make some brief comments on definitions and on the various behavioural models currently employed to investigate anxiety-related behaviours~ since these have a bearing on the interpretation of data. 1.1. ANXIETY: FORMSAND COMPONENTS Anxiety is an emotional state which is subjectively experienced as unpleasant or threatening. Symptoms include changes in mood (e.g. apprehension and fear) and cognitions (e.g. thoughts of impending mishap) and is generally accompanied by physiological and behavioural changes such as palpitations, sweating and hypervigilance (see Nutt, 1990). Unlike other psychiatric disorders, anxiety is a normal emotion serving as a useful arousal response to threatening or stressful stimuli, However, it becomes pathological when the response to the perceived stimuli becomes overexagerrated, irrational and disruptive and hence interfers with a person's ability to function normally. A distinction can be made between 'state' and 'trait' anxiety. 'State' anxiety is often associated with a particular stressful event and remits once the stimulus is removed. 'Trait' anxiety is a persistent feature of an individual that is part of their personality. In psychiatric practice a number of forms of pathological anxiety are currently considered to exist. The DSM-III-R (American Psychiatric Association, 1987) classification system distinguishes between the diagnostic classification of generalised anxiety-disorders, panic-disorder and phobia. Patients suffering from the former disorder generally score high on measures of 'trait' anxiety whereas those with simple phobias score low on 'state' anxiety unless a feared stimulus is presented. Whilst the DSM-III-R concepts are not fully accepted in the U.K. it is generally agreed that the different forms of anxiety respond to different pharmacological and therapeutic approaches. This suggests that the underlying neuropathology may differ in the different forms. As well as differing forms of anxiety-disorders, each form may be considered as having at least three components. LeDoux (1986) has proposed that these are (i) the subjective experience of anxiety, (ii) the evaluation procedure that leads to the generation of anxiety and (iii) the expression of anxiety. The neuroanatomical substrates underlying these different components are likely to be different. In attempts to identify the neuroanatomical basis of anxiety researchers should take into account the contribution played by these different components in their experimental models.

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1.2. THE USE OF ANIMALBEHAVIOURALMODELSTO ASSESSANXIETY Animal behavioural models of anxiety-disorders are unlikely to contribute significantly to our understanding of the neuroanatomical basis of the more subjective elements of normal and pathological anxiety. In contrast, brain imaging studies (see Section 5.2) in the living human brain are more likely to provide important information in this area. Nevertheless animal behavioural models have yielded much information on the neuranatomical structures and pathways involved in the expressive and evaluative components of anxiety. Whilst it is not the purpose of this review to evaluate the different models of anxiety, a few salient points should be noted. Animal tests of anxiety can be divided into several categories (see File, 1987; Weiss and Uhde, 1990; Lister, 1990). These include those based on conditioned fear or conflict, those in which anxiety is generated by novel environments and those in which anxiety is generated by pharmacological agents or by electrical stimulation of discrete brain areas. Conflict tests have been widely used. The paradigm involves repeated alternating exposure to two different experimental schedules. In the Geller-Seifter test rats are trained to press a lever to obtain a food reward. In the punished schedule the rats also receive an electric shock signalled by a light whereas in the unpunished schedule pressing for the food reward is not punished by electric shock. A similar protocol exists in the Vogel test but in this case thirsty rats are trained to drink from a spout. In both these tests it is presumed that the behavioural suppression (reduced response rate) that occurs in the punished 'conflict' component of the schedule is a result of the animal anticipating the punishment. Anxiolytic drugs such as the benzodiazepines increase rates of punished but not unpunished responding. In contrast to conflict tests, tests involving exposure to novel environments do not depend on the rat being conditioned to fear a stimulus that is linked to a primary drive behaviour such as eating or drinking. Instead, these ethologically based tests rely on the rodents innate fear of novel environments and measure natural behaviours. For example, the social interaction test (File, 1980) relies on the social behaviour of rats to decrease under aversive situations. In this test the factors that modify behaviour are the lighting level and the familiarity with the environment. Another test, the elevated plus or X maze (Handley and Methani, 1984; Pellow et al., 1985) is based upon the relative degree of exploration into the open and enclosed arms of the elevated maze. The rationale being that the open arms are more fear-provoking than the enclosed ones. The light-dark box exploits the rodents natural aversion to bright areas compared to darker ones. In this model animals make fewer crossings to the light side than the dark one (Crawley, 1981; Costall et al., 1987). In the aforementioned tests an anxiolytic drug would be expected to increase the behavioural responses to rodents in the most aversive environment. Whilst the ethologically based tests are attractive in the sense that they measure natural behaviour, results from these tests have in general produced conflicting results on the potential anxiolytic activity of pharmacological agents from various research laboratories. Perhaps this is not too surprising since in these tests, measurements of spontaneous behaviour are made in relatively uncontrolled environments in which variables such as apparatus design, animal handling, baseline activity may influence the results (see Green, 1991). In contrast to behavioural models which evoke behavioural inhibition, other tests produce an enhanced response. For example, in the fear-potentiated startle paradigm (see Davis, 1986, 1990) a conditioning stimulus (e.g. light) that is previously paired with foot shock increases the acoustic startle reflex response. Electrical stimulation of the periaqueductal gray is considered to be aversive (see Graeff, 1984) and in this model animals are conditioned to make a response (e.g. lever press) to avoid the aversive stimulus. Drug discrimination procedures have been employed to model chemically-induced anxiety (Lal and Emmett-Oglesby, 1983). This model uses a two-choice situation in which pressing on one lever will produce a food reward after a saline injection and pressing on the other will result in a food reward after injection of an anxiogenic drug. This model is considered to be a good model for revealing something about the subjective drug experience. It is clear from the aforementioned discussion that the different models measure different aspects of anxiety. Moreover, the components of anxiety (i.e. the subjective experience, the evaluation procedure and the expression of anxiety) are likely to differ amongst the models. For example, in

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some, such as the conflict tests, conditioning is an important component whereas in others, such as the plus-maze, animals have to make a decision about exploring areas with different levels of aversion. These different components will no doubt involve recruitment of different neuronal systems in the different models, although there are also likely to be common neuroanatomical structures and pathways involved. Given that the different models measure different forms of anxiety with a different neuronal basis, it is not surprising that clinically effective anxiolytics and those under investigation as potential anxiolytics, display different behavioural profiles in the different models. In considering the neuroanatomical basis of anxiety it is therefore important to take into account the anxiogenic stimulus that is presented, how this may be interpreted prior to making a response and also how other factors such as previously learned experiences influence the response. It is also worth commenting that many animal models are biased to model state anxiety and as such may be questioned for their usefulness in the development of drugs for the treatment of generalised anxiety-disorders. However, it is not easy to devise models for trait anxiety. One possibility is the use of genetically 'anxious' strains of animals, although as yet there has been little validation of the use of such strains.

2. NEURAL PATHWAYS INVOLVED IN ANXIETY-RELATED BEHAVIOURS: EVIDENCE FROM ELECTRICAL STIMULATION AND LESION STUDIES 2.1. HISTORICALPERSPECTIVE Up until the early part of this century emotion was considered to be determined or encoded peripherally. Central to the James-Lange theory of the late nineteenth century (James, 1884) was that emotions were only perceived after responses to a threatening stimulus had been made. That is we react to a threatening stimulus first and then feel the fear, or, we are happy because we laugh. Whilst this theory recognised that the perception of emotion occurred in the cerebral cortex it was considered to be a result of feedback from peripheral sensory receptors that had been activated as a result of the enhanced skeletal muscle and autonomic responses involved in producing the escape response. The experimental work of Cannon and of Bard using surgical ablation techniques resulted in this theory being seriously challenged in the 1920s. For example, Cannon (1927) amongst a variety of other evidence, disputing the James-Lange theory, noted that dogs are still capable of normal rage reactions even if the spinal cord is transected at the cervical level, thereby abolishing sensory receptor feedback. Another important objection to the James-Lange theory is that the reactivity of the visceral organs is generally too slow to be a direct cause of the conscious awareness of emotion. In 1929, Bard showed that in cats with the cerebral cortex and thalamus removed an emotional response termed 'sham rage' could occur together with autonomic responses such as increased heart rate. The term 'sham' was used because the behaviour was poorly directed and elicited by a variety of stimuli. When a surgical transection of the midbrain was made this emotional behaviour was lost. It was suggested that the hypothalamus is an important region for integrating the behavioural and autonomic expression of emotions and that the cortex serves to inhibit these defensive expressions. Some years later the neurologist James Papez (1937) made the statement "emotion is such an important function that its mechanism, whatever it is, should be placed on a structural basis". In a review based upon the known anatomy of the brain, findings from patients with damage to particular brain regions and from findings from these early ablation experiments, Papez (1937) proposed that the mammillary body of the hypothalamus is the site of output for the expressions of emotions. He suggested that sensory afferent information on arriving at the thalamus is split into three pathways: the pathway to the cortex representing 'the stream of thought'; the pathway to the basal ganglia representing the 'stream of movement' and the input to the hypothalamus representing the 'stream of feeling'. The input to the thalamus could be transmitted upstream towards the cortex or downstream to the brain stem and spinal cord. The downstream signals were important for eliciting the autonomic and behavioural reactions. Papez considered the mammillary body of the hypothalamus as important for sending projections to the cingulate cortex, which he

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Anterior thalamus

Cingulate cortex

Mammillary body

Association cortex

Hippocampus

Thalamus

Sensory input from environment

Signals to brain stem to elicit autonomic & behavioural reactions

FIG. 1. The model of emotion proposed by Papez (1937). considered to be the area in which the subjective emotional experience arises. Connections with other cortical areas would also be involved with emotional experience. Connections back to the hypothalamus via the hippocampus enable the hypothalamus to discharge downstream in the expression of emotion. Hence the hypothalamus can be activated in two ways: either by receiving sensory inputs directly from sensory pathways via the thalamus or by inputs from the cerebral cortex. In summary, the Papez circuit can be visualised as a means whereby the subjective experiences at the level of the cingulate gyrus can be combined with the emotional expression of the hypothalamic output. Hence emotional expression depends on the intergrative action of the hypothalamus whereas emotional experience requires participation of the cortex (see Fig. 1). More recent work supports the role of the hypothalamus proposed by Papez. In humans, electrolytic lesions of the posterior hypothalamus reduce anxiety to the extent that the procedure has been termed sedative surgery (see Kuhar, 1986). Furthermore, aggressive patients undergoing electrical stimulation of this area prior to lesioning reported an increase in heart rate and reported intense fear and anxiety (Schvarez, 1977). At about the same time as the Papez's circuit was published, Kluver and Bucy (1939) reported results of ablation of the temporal lobes in primates. This lesion which also destroys subcortical structures such as the amygdala evokes a complex behavioural syndrome including changes in sexual behaviour, feeding and emotional behaviour such that reactions associated with fear are reduced. Later studies confirmed that the amygdala had a prime role in this syndrome. It is interesting that Papez omitted the amygdala in his theory. Nevertheless, Maclean (1949) and others have extended Papez's theory to include the amygdala. The limbic system as we now know it has evolved from these early concepts that the Papez circuit, amygdala and other structures of the limbic lobe are important in the evaluation of stimuli and in the resulting experience and expression of emotions such as fear and anger. 2.2. COMPONENTS OF THE PAPEZ CIRCUIT The functional role of the mammillary body is little understood despite the widely accepted view of it being a component of the Papez circuit. Few studies have investigated anxiety-related

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behavioural responses following selective lesions of the mammillary body. Shibata et al. (1986) reported that electrolytic lesions of the mammillary body caused an increase in punished responding in a conflict test in the rat. This response did not develop however until 10 days after the lesion as compared to 5 days following lesions of the central amygdaloid nucleus. These data suggest that the behavioural disinhibition is probably a result of a slowly developing secondary degeneration in other brain structures either afferent or efferent to the mammillary body. More recently, this group (Yamashita et al., 1989b) have extended these studies to include electrolytic lesions of another structure of the Papez circuit, the anterior thalamic nucleus, and the dorsal tegmental nucleus which is intimately connected with the mammillary body. Anticonflict effects were absent following lesions of the anterior thalamus whereas lesions of the dorsal tegmental nucleus prolonged the latency to start drinking without influencing the number of shocks received. Based on these data and lesions of other structures, the authors conclude that the mammillary body, central amygdala, frontal cortex and septum have a key role in the mediation of conflict behaviour with the dorsal tegrnental nucleus having a facilitatory role. More recent studies have employed chemical lesioning techniques to produce lesions of discrete brain structures. Unlike electrolytic lesioning methods which destroy both cell bodies and fibres of passage, chemical lesions with substances such as ibotenic acid will only destroy the former. Laurie et al. (1990)* found no effect of ibotenic acid lesions of the mammillary body upon behaviour in the elevated plus-maze one week post lesion, suggesting a lack of involvement in the neural circuits generating anxiety in this paradigm. Taken together, these studies suggest a role for the mammillary body in the circuitry involved in behavioural suppression but not that involved in anxiety generated by the plus-maze. However, the responses to anxiolytic and anxiogenic drugs in the plus-maze can be attenuated following lesions of the mammillary body implicating an involvement of this structure in anxiety* (Laurie et al., 1990). Other suggested roles of the mammillary body include an involvement in memory processing. Disruptions of spatial memory in mice have been induced by lesions of the mammillary body (Tako et al., 1988). Such an effect might disrupt behaviour in the conflict test or the plus-maze paradigm. However, no disruption of behaviour was noted in the plus-maze* (Laurie et al., 1990). Moreover, memory tasks involving rewards or a natural means of escape from painful stimuli are not disrupted by electrolytic lesions of the cat mammillary body (Krieckhaus, 1967). Similarly in monkeys lesions of the mammillary body, unlike those of the hippocampus, do not produce long-lasting memory impairment (Zola-Morgan et aL, 1989). Nevertheless, the mammillary body may be important in some memory processing associated with anxiety-provoking situations.

2.3. AMYGDALA 2.3.1. Introduction The amygdaloid complex contains two divisions; the older corticomedial divisions and the newer basolateral area. Unlike other limbic structures it has a close relationship with the basal ganglia. One of the problems in studying the amygdala is that it is composed of a variety of separate nuclei each of which has irregular boundaries and is long and narrow. This creates problems when attempting to make discrete lesions or stimulate electrically without involving other nuclei. Moreover, investigations of the functional role of the different nuclei are complicated by fibres which pass through this area, interrelationships and connections with diverse brain structures. It is therefore not surprising that a variety of responses have been reported following lesions and stimulation of the amygdala (see Isaacson, 1982; Le Doux, 1986; Davis, 1990). Electrical stimulation of the amygdala produces effects on autonomic activity including changes in heart rate and respiration. Orientation, habituation and exploratory behaviours are also affected (Isaacson, 1982). Stimulation of the amygdala also evokes fear behaviours and defence reactions in animals. For example, Ursin and Kaada (1960) found a dissociation of fear (flight) and aggressive behaviour following stimulation of different parts of the amygdaloid complex in cats. Fight and flight could *Laurie, D. J. and Pratt, J. A. Mammillary body lesions attenuate the anxiogenic action of FG 7142 in the elevated plus-maze. Pharmac. Biochern. Behav., submitted for publication.

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be obtained from the rostral regions such as the lateral and central nucleus. Defence or aggressive reactions could be obtained from the medial and caudal regions. Interestingly, the areas where flight and defensive behaviours could be evoked did not correspond to the established nuclear division of the amygdala. In general, animals with amygdaloid lesions display low social dominance when compared to nonlesioned animals. This effect may be related to a reduced ability to respond to social signals and may explain why they respond in an apparently fearless manner when placed in a threatening situation (see Isaacson, 1982). In humans stimulation of the amygdala evokes feelings of fear (see Kuhar, 1986; Chapman et al., 1954).

2.3.2. Amygdala and Learned Behaviours The effects of amygdaloid lesions have been studied in many types of learning situations that involve fear or fear motivation. In general results from lesion studies do not support a role for the amydala in learning and memory per se although electrical stimulation studies suggest an impaired retention of learned performances after amygdala stimulation. Nevertheless, lesions of the amygdala do influence performance in certain fear-motivated test situations such that the initiation and continuation of behavioural acts is impaired. For example, in the two-way active avoidance task amygdala lesions produced a deficit in terms of the number of trials required to begin to make avoidance responses. However, once animals began to respond learning progressed at the same rate as in control animals. The formation of the conditioned emotional response is also disrupted following lesions of the amygdala. The emotional reaction is measured by the degree to which operant responding is suppressed during the presentation of a stimulus (light or sound) that was previously associated with an aversive stimulus such as foot shock. It is generally thought that the animals become afraid of the signal and that this fear results in the lowered rate of bar pressing for food or water. The loss of the response in the lesioned animals does not, however, mean that the animals are less afraid of the stimulus. One alternative explanation could be that they have an impaired ability to suppress ongoing behaviours (see Isaacson, 1982). There is much discussion of the functional role of the different amygdaloid nuclei. The central nucleus has been implicated in different types of conditioning. Often conflicting results have appeared in the literature. This seems to depend in part upon the type of lesion employed. Electrolytic lesions have the disadvantage of destroying fibres of passage as well as destroying neurons in the vicinity. Hence it is difficult to dissociate between an effect on local neurons or on fibres originating in other areas. Chemical lesions with ibotenic acid will only destroy neurons in the vicinity. Studies by Riolobos and Garcia (1987) showed that electrical lesions but not those produced by ibotenic acid produced a deficit in acquisition of passive avoidance conditioning suggesting that the lesion is caused by lesions of fibres crossing the central amygdaloid nuclei rather than intrinsic neurons in the nucleus. In contrast Jellestad and Bakke (1985) found that both types of lesions impaired acquisition of passive avoidance responses. However, the lesion involved damage to the central, lateral and basolateral nuclei thus making it impossible to implicate any one nucleus in the behavioural deficit. In a later study Jellestad et al. (1986) selectively destroyed the central amygdala using ibotenic acid and found an increase in activity in the passive avoidance task. Electrolytic lesions produced delays in passive avoidance responses supporting the view that the behavioural deficit is dependent on fibres of passage passing through the central amygdala. Similarly, Grossman et al. (1975) reported that lesions of the central and basolateral but not other amygdaloid nuclei suppressed the acquisition of passive avoidance.

2.3.3. The Amygdala as Part of the Circuitry Involved in Conditioned Fear The extensive studies of LeDoux (1986) and of Davis (1986, 1990, 1992) and colleagues have yielded important information on the neuronal circuitry involved in conditioned fear. LeDoux (1986) has reviewed the results of lesion studies aimed at identifying the circuits necessary for the expression of conditioned fear in response to acoustic stimuli. The model used typically involves pairing an auditory tone with foot shock. The expression of conditioned fear is characterised by changes in autonomic activity (increases in arterial pressure) and freezing behaviour. Both JPT55/2--E

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responses are abolished by lesions of two auditory structures; the inferior colliculus and the medial geniculate nucleus of thalamus but not by lesions of the auditory cortex. The auditory signals would thus appear to be transmitted directly from the auditory thalamic region to the amygdala (Le Doux, 1986). Of the amygdaloid nuclei, LeDoux et al. (1988) propose that the central nucleus is likely to be the output structure whereas the lateral and/or amygdalostriatal region may serve as the sensory interface of the amygdala during conditioning. If the central nucleus is an important link in the circuitry mediating the autonomic and behavioural responses of conditioned fear then lesions of efferent target structures should abolish such responses. Ibotenic acid lesions of the midbrain central gray region disrupted conditioned 'freezing' responses but not the conditioned changes in blood pressure in rats. Conversely, lesions of the lateral hypothalamic area blocked the cardiovascular but not the behavioural response. The lateral hypothalamus is known to project to brain stem autonomic centers such as the nucleus tractus solitarius and the dorsal motor nucleus of vagus. Lesions of another efferent structure, the bed nucleus of stria terminalis, was without effect on either response. Thus it would appear that different efferent projections of the central amygdala mediate the behavioural and autonomic correlates of conditioned fear. Since lesions of the central amydala itself or earlier auditory structures disrupt both behavioural and autonomic responses, this would suggest that the pathways mediating these responses diverge after the amygdala (LeDoux et al., 1988). These findings support those of Smith et al. (1980) which showed that the electrolytic lesion of the lateral hypothalamus disrupted the autonomic expression but not the behavioural conditioned emotional response in primates. The extent to which these behaviours can be correlated with human anxiety is however unclear. 2.3.4. The Amygdala, Fear Potentiated Startle and Other Models of Anxiety An animal model that has been proposed by Davis (1990, 1992) to be more relevant to human fear and anxiety is the fear potentiated startle paradigm which can be loosely connected with human anxiety on the basis that people startle more when they are afraid. Davis argues that this model, unlike many others (e.g. conflict test, social interaction test and elevated plus maze test), does not rely upon the measurement of fear as a cessation or inhibition of behaviour. Instead, in the startle model the amplitude of the startle reflex is enhanced when it is evoked in the presence of a conditioned stimulus (e.g. visual or auditory stimulus) that was previously paired with a shock. Davis (1990, 1992) suggests that models that do not rely upon response inhibition to measure a state of fear or anxiety may detect different types of anxiolytics as compared to models which activate the circuitry involved in behavioural inhibition. He hypothesises that common neural circuits are involved in the behavioural inhibition evoked by the various models that measure suppression of behaviour. Hence drugs which act upon the circuitry involved in behavioural inhibition may have similar effects in each of these tests. Such an action might not involve neurons involved in the perception or experience of anxiety. However, it should be noted that whilst the startle paradigm does appear to activate different neural systems to those involved in conflict paradigms, the amygdala is a common structure of recruitment (see Figs 2 and 3). At present it is unknown whether similar neuronal systems are activated in other models involving suppression of behaviour such as the plus-maze and social interaction test. Indeed, the differing pharmacological profiles of various anxiolytics in these ethologically based tests (see Chopin and Briley, 1987; Green, 1991) would suggest actions in separate neural systems. The neuronal systems involved in the fear potentiated startle paradigm have been intensively studied by Davis and colleagues using lesioning and electrical stimulation studies (see Davis, 1990, 1992). They have shown that the acoustic startle reflex is mediated by the ventral cochlear nucleus which projects to an area close to the ventral nucleus of the lateral lemnicus and then to an area dorsal to the superior olives, the nucleus reticularis pontis caudalis. Hence electrical stimulation of each of these nuclei will evoke a startle-response with progressively shorter latencies as the electrode is moved down the pathway. Davis and colleagues propose that the central nucleus of amygdala is critically involved in the fear potentiated startle response. Hence lesions of this structure completely eliminate potentiated startle (Hitchcock and Davis, 1986) whereas lesions of structures thought to be important in motor conditioning such as the cerebellum and the red

The neuroanatomical basis of anxiety

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FIG.2. Diagram of proposed neuronal circuits involved in behavioural inhibition as implicated from brain lesioning studies in conjunction with conflict tests. BI, basolateral and C, central amygdala. nucleus were without effect. In accordance with these results electrical stimulation of the amygdala enhances acoustic startle (Rosen and Davis, 1988). It is proposed that when a visual conditioning stimulus is employed the central nucleus of amygdala becomes activated via the lateral geniculate and perirhinal cortex. Projections from this amygdaloid nucleus to the startle pathway are proposed to occur at the level of the reticularis pontis caudalis (see Fig. 3). It now seems clear that the projections of the central nucleus of amygdala with a variety of brain stem structures are important in the expressions of the symptoms of fear and anxiety (LeDoux, 1986; Davis, 1990, 1992) (see Fig. 4). Thus projections from the lateral hypothalamus or to the dorsal motor nucleus of vagus are involved in several autonomic measures of fear or anxiety such as changes in heart rate and blood pressure. Projections to the trigeminal and facial motor nuclei may be involved in the facial expressions of fear. Projections to the central gray may mediate the suppression of behaviour observed in tests such as conflict and social interaction. The connections with the ventral tegmental area may mediate stress induced changes in dopamine turnover in the frontal cortex and this connection may also link in the locus coeruleus that has been implicated in fear and anxiety (Redmond and Huang, 1979) and in attention and vigilance (Aston-Jones and Bloom, 1981; Aston-Jones et al., 1990). In addition to its clear involvement in the fear potentiated startle-response, the amygdala has also been implicated in other animal models of anxiety. In the water lick conflict test, electrical lesions of the anterior part of the basolateral amygdala, the central amygdala but not the medial amygdala produced an anticonflict effect (Shibata et al., 1989). In contrast the results of Hodges et al. (1987) suggest that the lateral/basolateral amygdala is of prime importance in conflict behaviour as injection of benzodiazepines into this region evoked an anticonflict effect. However, a recent report showed that chlordiazepoxide still had a significant anticonflict effect after amygdala lesions (Yadin et al., 1991). These findings do not exclude a role for the amygdala in behavioural suppression but merely suggest that it is not as critical as its involvement in fear potentiated startle. The accumulating evidence that lesions of various interconnected limbic structures can modify

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Proposed neural pathways involved in fear-potentiated startle using a visual conditioned stimulus. Inputs both from the retina, via a projection involving the ventral lateral geniculate nucleus and the perirhinal cortex, and from pain afferents in the spinal cord, may converge at the lateral and basal nuclei of the amygdala. After being paired with a shock, the light may activate the lateral and basal amygdaloid nuclei, which in turn project to the central amygdaloid nucleus. Activation of the central nucleus of the arnygdala may be both necessary and sufficient to facilitate startle through a direct connection to the nucleus reticularle pontis caudalis, an obligatory part of the acoustic startle pathway.

FIG. 3. Reprinted from Davis (1992) with permission of the author and the copyright holder, Elsevier Scientific Publishing Co., Amsterdam. conflict behaviour would suggest that this behaviour is dependent on integrated activity in a variety of structures rather than being mediated by a single structure (see Fig. 2, Sections 2.2-2.6). 2.4. PERIAQUEDUCTALGRAY The dorsal midbrain central gray (or periaqueductal gray, PAG) deserves mention since it has been considered along with the amygdala and medial hypothalamus to be involved in aversive behaviours. Electrical stimulation of the PAG evokes fear and aversion expressed in animals by flight behaviours and defensive reactions including aggression (Olds and Olds, 1963). These are accompanied by autonomic changes including a rise in blood pressure. Similarly, in humans undergoing neurosurgery, stimulation of the PAG elicited unpleasant and fearful sensations whereas a small lesion of this area resulted in marked calming of patients undergoing suffering (Nashold et al., 1969). The notion that aversive motivational states are generated by electrical stimulation of the PAG is supported by the fact that animals will readily learn to make a behavioural response to switch off the electrical stimulation. Often this is achieved by the brain stimulation automatically being switched off (escape) when the animal crosses the midline that separates the two compartments of a shuttle-box. Benzodiazepines, ethanol and barbiturates significantly raise the thresholds of these operant escape reactions and this has led Graeff (1984) to propose that minor tranquillisers depress the functioning of the brain aversive system (comprising the PAG, medial hypothalamus and parts of

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~. lateral hypothalamus /

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~, dental motor nucleus of vagus nucleus ambiguous ~, parabrachial nucleus ~ ventral tegmentsl area Ionua coeruleus dorsal lateral tsgmentsl nucleus nuciaus of

/ u nco~..tnio.nld ~ " fear idlmulul

\

sau,,le

- central grey trigeminal,feclal motor nucleus ~ paravantrlculer nucleus

effect ~, sympathetic activation

l, parasympathetic activation D, increased respiration p activation of dobemine, nompinel~rlne and acelylcholine ~, incraal~l reflexes t. cessation of behavior mouth open, jaw movements P ACTH release

159

llNit Or l l l r l of fear or anxiety ~. tschycardia. Galvanic skin response paleness, pupil dilation blood pressure elevation ulcers, urination, defecation bradycardia p panting, resl~ratcry distress m, behavioral and EEG arousal increased vigilance P increased startle p freezing, conflict test, CER social interaction facial expres.~ons of fear ~, corUco6tsrcid release (stress response)

Dtagram of direct ~ ~ the c . w ~ tluclsuI of the l f ~ d l i I artd li variety of ~ areas that may be involved in diffemnt animal tes~ of feu a~d ~ - ~ , . ~ oondl~oned ernotional m~tx~ae.

and bcainMern B ~

FIG. 4. Reprinted from Davis (1992) with permission of the author and the copyright holder, Elsevier Scientific Publishing Co., Amsterdam. the amygdala) and that this action could mediate at least part of their clinical anxiolytic effect. Whilst Graeff (1984) acknowledges that other neuronal systems such as the septohippocampal formation (see Section 2.5) are important in the actions of anxiolytic drugs, he argues that such drugs would also be expected to possess antiaversive activity (and not just disinhibit behaviour) since anxiety is an unpleasant sensation which has strong aversive motivational properties. In contrast (as discussed in Section 2.3), others argue that projections from the amygdala to the central gray are important in the expressions of the symptoms of fear and anxiety (see LeDoux, 1986; Davis, 1990), such as freezing responses and may mediate the suppression of behaviour observed in conflict and other behavioural tests. Indeed, the initial response to PAG stimulation is to freeze but it is the learned escape-response that is the behavioural response monitored. It may also be important to take into account the fact that both the median and dorsal raphe project to the PAG (Beitz et aL, 1986). Interestingly, electrical stimulation of median raphe produced behavioural inhibition and signs of fear in the rat (Graeff and Silveira Filho, 1978) which was considered to be a result of altered 5-hydroxytryptamine (5-HT) inputs to the septohippocampal formation. However, the role of 5-HT inputs to the PAG in relation to aversive behaviour requires clarification. The clear involvement of 5-HT in pain at the level of the PAG may suggest that alterations in pain processing cannot be excluded from behaviours evoked by PAG stimulation. 2.5. SEPTOHIPPOCAMPALSYSTEM

The involvement of the septohippocampal system, together with ascending monoamine projection pathways in anxiety, has been expounded by Gray (1987). In this model, it is proposed that the septohippocampal system is the neuroanatomical basis of a 'behavioural inhibition system' and that the noradrenergic projections from the locus coeruleus and the 5-HT projections from the raphe to the hippocampal formation may modulate the sensitivity of this system. With regard to the locus coeruleus, Gray proposes that its role is as a 'general alerting and alarm system' whereby certain stimuli that enter the septohippocampal system from the entorhinal cortex are subjected to particularly careful checking. The Papez circuit has also been incorporated into the model; it is envisaged that this, together with the septohippocampal system and the entorhinal area, serve to allow the system to operate effectively as a whole by predicting and checking sensory events and effecting appropriate outputs. The behavioural inhibition system is just one of three emotional systems proposed by Gray (1987), the other two being a fight/flight system which mediates the behavioural responses (e.g. defensive aggression) of unconditioned punishment and nonreward, and an approach system which

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J.A. PRATT

mediates the behavioural responses (e.g. active avoidance) to secondary appetitive stimuli that are paired with reward or nonpunishment. Gray suggests that anxiety-producing stimuli that activate the behavioural inhibition system are those associated with conditioned punishment and nonreward together with novelty and innate fear. The septohippocampal system is proposed to function as a comparator to evaluate whether such incoming stimuli are anxiety-provoking and if so, a variety of behavioural responses are produced. These responses include inhibition of ongoing behaviours whether classically conditioned or innate (e.g. exploration, feeding) increased attention and increased arousal. It is of interest that behavioural inhibition and increased arousal may occur simultaneously. For example, animals exposed to occasional nonreward hesitate more (behavioural inhibition) and run faster (higher arousal) than continuously rewarded animals. Most of the evidence to support Gray's theory of anxiety has arisen from lesion studies of the septohippocampal formation. Gray and colleagues have investigated the effects of lesions of the septum and hippocampus on behaviour that they suggest to be a reflection of anxiety-related processes. These include behavioural tests that measure resistance to the effects of punishment and extinction. For example, if the reinforced behaviour of an animal is infrequently punished during training, then there will be less suppression of behaviour (i.e. resistance to punishment) when the animal is tested later and punished for every response, than if punishment had never been administered. Lesions of the lateral septum abolish this differential effect, i.e. the response in the test situation is similar, despite a different punishment protocol during training. In contrast, medial septal lesions increase responding and the different effects of previous training still emerge. Lesions of the hippocampus produce effects similar to the combined lesions. Based on these and other results and a knowledge of the neuroanatomy, Gray postulates that the hippocampus receives signals of nonreward or punishment from the medial septum. This activates the 'behavioural inhibition system'. Hence, on evaluating the situation the hippocampus will adjust its output (via the subiculum) by signalling to the lateral septal nuclei which in turn then alters the input of the medial septal nuclei to the hippocampus, thereby attentuating the normal inhibition of nonrewarded behaviour. This hypothesis explains the findings that lesions of medial septal nucleus prevent behavioural inhibition and an increase responding during extinction or punishment irrespective of training without interfering with the effect of training. Lesions of the lateral septal nucleus result in an inability of the system to use the information obtained by the prior training history. Gray (1987) also reports that benzodiazepines produce similar effects to those of septohippocampal lesions in these behavioural paradims. He argues that benzodiazepines impair processing of aversive stimuli and thus inhibit responding that is normally suppressed by a septohippocampal behavioural 'inhibition system'. It should be borne in mind, however, that the septohippocampal system is involved in a variety of other behavioural processes and in particular in learning and memory. Such effects may explain the results obtained with benzodiazepines that have, in addition to anxiolytic effects, memory impairing properties. Whilst it is unclear at present the relevance of the behavioural paradigm employed by Gray to human anxiety, he postulates that in man the activation of the behavioural inhibition system would be identified as experienced fear, anxiety, or disappointment, depending on the environmental circumstances. However, a system such as behavioural inhibition which is concerned with the evaluation of aversive stimuli may not only be involved in the manifestions of anxiety but have wide-ranging functions in terms of learning to cope with environmental changes. Nevertheless, other groups have shown an involvement of the septum in more widely used behavioural models of anxiety. For example, lesions of the septum produce antianxiety effects in the elevated plus maze test and the shock probe burying test (Treit and Pesold, 1990). Similarly, Yamashita et al. (1989b) found that lesions of the septum produce a modest increase in punished responding in the Vogel type conflict test in rats, although this was not as pronounced as lesions of the mammillary body, central amygdala and frontal cortex. Lesions of the dorsal hippocampus did not modify behaviour in a conflict test (Yamashita et al., 1989b) but this may have been because an inappropriate area of the hippocampus was lesioned. 2.6. RAPHENUCLEI Neurons containing 5-HT are primarily confined to defined raphe nuclei in the brain stem (Azmitia and Segal, 1978; Parent et al., 1981; Kosofsky and Molliver, 1987). From these groups

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of nerve cell bodies there are ascending projections to numerous forebrain areas. Projections from the dorsal raphe innervate the nigro-striatal system primarily, but also project to the cortex (mainly frontal), the molecular layer of the dentate gyrus and to amygdaloid nuclei. In contrast, there is a more widespread innervation of the limbic/cortex system from the median raphe. These include most areas of the neocortex, amygdaloid nuclei, cingulate cortex and several fields of the hippocampus. The morphology of the axon terminals arising from the two nuclei also differ; small varicose axons arise exclusively from the dorsal raphe while large varicose axons arise only from median raphe (Kosofsky and Molliver, 1987). Whilst 5-HT pathways have been implicated in anxiety since the early 1970s, there still exists controversy in the literature. This has arisen partly because the different 5-HT pathways may have different functional roles and also because of the recent advances in knowledge regarding the presence of multiple types of 5-HT receptors in brain. The neurotoxin 5,7-dihydroxytryptamine (DHT) has been employed to induce relatively selective lesions of 5-HT pathways in the brain. In the conflict test the intraventricular administration of 5,7-DHT is reported to evoke a release of punished responding or to produce no effect. These differences may relate to the differences in 5-HT availability at different points in time subsequent to the lesion (see Iversen, 1984; Kahn et al., 1988). Such studies do not however, distinguish between the effects on the different 5-HT pathways as a generalised decrease in 5-HT function occurs. More selective lesions produced by injection of 5,7-DHT into the ventral tegmentum resulted in a marked release of punished responding in a Geller-Seifter conflict test (Tye et al., 1977). The changes were considered to be attributable to an action in 5-HT pathways innervating limbic structures and the cortex as this lesion left the innervation of the striatum relatively intact. The effect could not be attributed to a nonspecific stimulation of motor function as there was no change in lever pressing for food during other components of the schedule. In support of this, electrical stimulation of the median raphe nuclei suppressed ongoing positively reinforced operant behaviour and was accompanied by somatic and autonomic signs of emotional behaviour (crouching, defaecation and piloerection) (Graeff and Silveira Filho, 1978). A later study reported that electrical stimulation of the median raphe or a threatening conditioning stimulus evoked similar behavioural and EEG responses (Graeff et al., 1980). The authors conclude that their results support the view that the median raphe projection to the septal area and hippocampus is a substrate of behavioural inhibition in the rat. In support of this, electrophysiological studies have shown that the firing rates of pyramidal cells in the hippocampus are inhibited by electrical stimulation of the median raphe as well as by local iontophoretic application of 5-HT (Segal, 1975). Gray (1987) subsequently suggests that a reduced 5-HT release in the hippocampus may be a mechanism by which benzodiazepine anxiolytics reduce the behavioural responses to aversive stimuli. In contrast, Thiebot et al. (1983) suggested that the dorsal raphe--substantia nigra pathway is important in behavioural inhibition. In a schedule involving shock-induced suppression of drinking, lesions of the dorsal raphe to the substantia nigra produced an attenuation of the behavioural inhibition. It was proposed that the nigral 5-HT innervation may be important for the control of motor components of behaviour and in particular those associated with the expression of fear and anxiety through the inhibition of ongoing responding, rather than any important features of the emotional state of anxiety. Thiebot et al. (1984a) also exclude the possibility that dorsal raphe projections to other forebrain structures such as the amygdala and accumbens are involved since destroying 5-HT innervation in these structures did not release punished behaviour. However, if 5-HT inputs to dopamine neurons in the basal ganglia are essential for response suppression then lesions of forebrain dopamine systems should disrupt behavioural inhibition. This does not appear to be the case (see Iversen, 1984). The forebrain projections from the raphe have also been implicated in ethologically based models of anxiety (see Table 1). Thus File et al. (1979) reported an anxiolytic effect of 5,7-DHT lesions of the dorsal raphe nuclei in the social interaction test. However, 5,7-DHT injections into both the dorsal and median raphe decreased social interaction (File and Deakin, 1980). More recently, Briley et al. (1990) have shown that intraventricular administration of 5,7-DHT produced an anxiolytic effect in the elevated plus-maze test. In contrast, Critchley and

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TABLE1. Effects o f Chemical Lesions o f Brain Structures on Behavioural Responses in Ethologically Based Models o f Anxiety

Brain Structure(s)

Response Social Interaction Plus-maze

Dorsal raphe Median raphe

Anxiolytic No effect

N.D.

File et al., 1979

Dorsal and median raphe

Anxiogenic

N.D.

File and Deakin, 1980

Dorsal raphe

N.D.

No effect

Critchley and Handley, 1989

Median raphe

N.D.

Anxiogenic

Balfour et al., 1986

All 5-HT pathways

N.D.

Anxiolytic

Briley et al., 1990

Septum

N.D.

Anxiolytic

Treit and Pesold, 1990

Mammillary body

N.D.

No effect

Laurie et al., 1990

Locus coeruleus

No effect

N.D.

Crow et al., 1978

Reference

N.D., not determined. Note responses refer to baseline responses. In some studies (Critchley and Handley, 1989; Laurie et al., 1990) responses to 5HT1A agonists and benzodiazepines were modified following lesions of the structures indicated. Handley (1989) reported that 5-HT lesions of the dorsal raphe did not influence baseline behaviour in this model although the anxiolytic responses to a 5-HT1A agonist were affected. In another study, injection of 5,7-DHT into the fimbria and cingulum bundle in order to destroy the 5-HT pathway innervating the hippocampus from the median raphe produced an anxiogenic response in the elevated plus-maze (Balfour et al., 1986). In summary, some but not all studies suggest a role of ascending 5-HT pathways in anxiety-related behaviour. However, the precise neuronal systems involved and whether the resultant behaviours are a reflection of the internal emotional state of anxiety requires further investigation.

2.7. Locus COERULEUS 2.7.1. Electrical Stimulation and Lesion Studies The noradrenergic neurons of the dorsal noradrenergic bundle that project from the locus coeruleus to the neocortex and hippocampus, as well as to the spinal cord, have been implicated in a variety of functions including arousal, learning, blood pressure control, stress and anxiety (see Mason and Fibiger, 1979; Robbins, 1984; Aston-Jones et al., 1990). A role for the locus coeruleus in anxiety was first suggested by Redmond and colleagues. Electrical stimulation of the locus coeruleus results in increased fear response in monkeys (Redmond et al., 1976). Conversely, primates with electrical lesions of the locus coeruleus are placid. There is contradictory evidence regarding similar effects in humans (see Mason and Fibiger, 1979). Pharmacological manipulation of this nucleus also appears to support a role of noradrenergic neurons in anxiety. In the locus coeruleus the firing rate of noradrenaline containing cells is under powerful inhibitory control by ct2-adrenoceptors located on the cell soma and dendrites. The ~2-adrenoceptor antagonist yohimbine increases locus coeruleus cell firing and evokes an increase in fear-associated behaviour in monkeys and can produce anxiety in humans. In contrast the ct2-agonist clonidine, which decreases locus coeruleus cell firing, reduces the incidence of fear behaviour (see Mason and Fibiger, 1979; Nutt, 1990). Fibiger and Mason (1978) argue that in fear motivated tasks such as passive avoidance, lesions of noradrenergic neurons would be expected to impair performance. Moreover, if this system is also involved in learned responses then lesioned rats should also be slower to learn the avoidance tasks. These authors could find no support for their hypothesis of a reduced fear following noradrenaline depletion in a variety of passive avoidance paradigms, although resistance to extinction was observed. In the social interaction test of anxiety 6-hydroxydopamine lesions of the

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locus coeruleus also fail to affect behaviour under any light/familiarity condition (Crow et al., 1978). Despite this lack of effect in some paradigms, lesions of the locus coeruleus do appear to influence neophobia in a number of other test situations. For example, in a maze apparatus novel to the rats, lesioned animals took longer to leave the start box and took longer to consume food pellets found in the unfamiliar goal box than controls (Mason and Fibiger, 1977). Hence these results would tend to suggest an increase in fear response, an effect opposite to that reported from the studies in monkeys. An alternative explanation is that noradrenaline lesions result in a failure to habituate to novel stimuli. This may account for the resistance to extinction observed in some studies (Mason and Iversen, 1979).

2.7.2. Responses of Noradrenergic Neurons to Stressful Stimuli Other evidence supporting a role of noradrenaline neurons in fear/anxiety responses is that locus coeruleus firing rates increase following stressful/threatening stimuli in both the monkey and cat (Abercrombie and Jacobs, 1987; Grant and Redmond, 1984). Moreover, stressors such as shock and immobilization are known to increase noradrenaline turnover in forebrain and benzodiazepines are known to prevent this (Corrodi et aL, 1971). Weiss et al. (1982) suggest that the increased firing of the locus coeruleus neurons may underlie the behavioural impairment following repeated exposure to unpredictable inescapable electroshock, an animal model more usually considered to be related to depression. They propose that the stressor evokes a massive release of noradrenaline such that the functional pool is lost temporarily. This would result in an understimulation of presynaptic ~2-autoreceptors and an increase in activity of locus coeruleus neurons. A role of the locus coeruleus in sensory processing capabilities has also been advocated. Aston-Jones and Bloom (1981) demonstrated an increase of firing of locus coeruleus neurons during exposure to a variety of non-noxious sensory stimuli. The magnitude of the bursting response was correlated with the state of vigilance of the animals (Foote et aL, 1980). Electrical stimulation of the locus coeruleus or microiontophoretically applied noradrenaline onto hippocampal pyramidal cells enhanced the inhibitory effect of an auditory tone on hippocampal firing (Segal and Bloom, 1976). Lesions of the locus coeruleus do not, however, produce sensory impairment. Robbins (1984) has attempted to interrelate some of the various hypotheses concerning the various functions of the locus coeruleus. Robbins proposes that the dorsal noradrenergic bundle does not itself mediate stress but becomes active during stressful situations. This results in a high state of arousal so as to maintain selective attention. These ideas are not too far removed from Gray (1987) who believes that this system is important for identifying certain stimuli that enter the septohippocampal system from the entorhinal cortex that require particularly careful checking. In other words, it acts as a general alerting and alarm system.

2.7.3. Noradrenergic Function in Patients with Anxiety Disorders Anxious patients, in particular those with panic disorder, display abnormalities in noradrenergic function. Such patients display hypersensitive responses to yohimbine in that there are marked increases in anxiety levels compared to healthy controls. The behavioural, endocrine and cardiovascular responses to the cq-agonist clonidine also differ from healthy controls. Clonidine evokes less sedation and a smaller increase in plasma growth hormone in patients with panic disorder but an enhanced response to the clonidine-evoked decreases in blood pressure, plasma metabolites of noradrenaline and cortisol. Interestingly, the decrease in anxiety, ratings are unchanged compared to controls (see Heninger and Charney, 1988; Nutt and Glue, 1989). It is difficult to suggest a simple hypothesis regarding an abnormality in the ~2-adrenergic system in patients with panic disorder as increased responses to both an ~z-antagonist and agonist occur. It may be that some responses could be attributable to alterations in postsynaptic ctz-receptors, whereas others are due to altered presynaptic 0t2-receptor function (see Nutt, 1990).

164

J.A. PRATT 3. PHARMACOLOGICAL MANIPULATION OF 5-HT SYSTEMS AND ANXIETY 3 . 1 . 5 - H T ANTAGONISTS AND REDUCTION OF 5 - H T FUNCTION

Using nonselective 5-HT antagonists, such as methysergide, cyproheptadine and methergoline, no consistent anxiolytic effect has been observed in conflict tests or ethologically based tests (see Chopin and Briley, 1987; Kahn et al., 1988). In contrast, depletion of 5-HT in the brain with parachlorophenylalanine (PCPA) has produced more consistent anxiolytic effects in several tests including conflict procedures and social interaction (see Iversen, 1984; Kahn et al., 1988). However, the results from the conflict procedure may be complicated by the fact that PCPA alters appetitive behaviour and so may not be reflecting an anxiolytic effect. 3.2. ENHANCEMENT OF 5 - H T FUNCTION

If decreasing 5-HT function evokes responses consistent with an anxiolytic effect then enhancing 5-HT function would be expected to increase anxiety. Unfortunately agonists such as mchlorophenylpiperazine (rnCPPP) and 5-methoxy,N,N-dimethyltryptamine (5-MeODMT) and 5-HT uptake inhibitors produce alterations in motor activity making it difficult to evaluate specific effects on anxiety. Compounds which prevent the uptake of 5-HT do not generally produce anxiolytic effects in behavioural tests (see Chopin and Briley, 1987). 3.3.5-HT RECEPTOR SUBTYPE-SPECIFIC LIGANDS The recent development of ligands selective for 5-HT receptor subtypes has led to intensive research into the role of 5-HT receptors and neuronal systems in anxiety (see Marsden et al., 1989; Chopin and Briley, 1987). A variety of 5-HT receptor subtype-specific ligands have been shown to possess anxiolytic activity in animal tests of anxiety. 5-HT~A receptor agonists such as buspirone and ipsapirone demonstrate anxiolytic effects in the clinic. However, the anxiolytic activity in behavioural tests has not been consistently reported by all groups (see Chopin and Briley, 1987). There is also evidence that the 5-HT 2 receptor antagonists, such as ritanserin, possess anxiolytic properties in a variety of animal tests of anxiety, albeit over a limited dose range (Critchley and Handley, 1987; Chopin and Briley, 1987). More recently, the 5-HT3 receptor antagonists have been shown to be active in certain animal tests of anxiety (Jones et al., 1988), although not all groups find these compounds to be active (File and Johnston, 1989). Consistent with a role of 5-HT 3 receptors in anxiety is that 5-HT 3 agonists such as 2-methyl 5-HT and 1-(m-chlorophenyl)-biguanide (mCPBG) are reported to be anxiogenic in the social interaction test (Costall et al., 1989; Mitchell et al., 1991). Despite the inconsistencies in the evidence, there is a growing consensus that a variety of 5-HT receptor agonists and antagonists have anxiolytic properties, strengthening the view of the role of 5-HT in anxiety. Some explanations given for the contradictory results found in behavioural tests are that the animal models used to detect anxiolytics may be particularly sensitive to benzodiazepine anxiolytics and are less sensitive to other types of anxiolytics, or, that they detect different forms and components of anxiety. The neuroanatomical basis of action of these ligands in anxiety remains to be fully clarified. Nevertheless it is generally agreed that 5-HT~A agonists produce their effects through an action on presynaptic somatodendritic autoreceptors of the raphe nuclei. Activation of these receptors leads to a reduction in raphe firing which in turn is thought to lead to a decrease in the release of 5-HT in the frontal cortex and hippocampus (see Traber and Glaser, 1987). Electrophysiological studies also indicate that these compounds have inhibitory effects on 5-HTtA receptors located on the hippocampal pyramidal cells (Martin and Mason, 1987; Sprouse and Aghajanian, 1988). It is of interest that benzodiazepines can also reduce the firing of raphe neurons (Gallager, 1978) raising the possibility that this is the common neuronal pathway important in the anxiolytic actions of these two distinct classes of compounds. The neuroanatomical basis of action of 5-HT2 antagonists in anxiety requires evaluation. Autoradiographic studies demonstrate a high density of postsynaptic sites in the cortex and olfactory tubercules, followed by the basal ganglia and lower densities in the hippocampus and

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165

cerebellum (Hoyer et al., 1986). The exact sites with which these drugs mediate their putative anxiolytic effects remains to be determined. With respect to 5-HT 3 antagonists there is some evidence for an action at the levels of the raphe nuclei and the amygdala (see Section 4.2). In summary, a common feature of anxiolytics such as benzodiazepines, 5-HT~A agonists and 5-HT2 antagonists, is a reduction in 5-HT neurotransmission. Future studies could usefully investigate whether specific changes in neuronal activity in discrete brain structures underlie the behavioural responses to these drugs in vivo. 3.4. INTERACTION BETWEEN BENZODIAZEPINES AND 5-HT SYSTEMS

The notion that benzodiazepines may alter 5-HT neurotransmission and that this may be relevant to their anxiolytic effects arose initially from the findings that manipulating the 5-HT system mimics the antipunishment effect of benzodiazepines. Biochemical studies have also demonstrated that benzodiazepines reduce 5-HT transmission. Biochemical indices of 5-HT nerve activity include measures of 5-HT turnover, 5-HT synthesis and [3H]-5-HT release. Experimental evidence shows that benzodiazepines decrease 5-HT turnover (Wise et al., 1972; Pratt et al., 1979, 1985; Collinge et al., 1983) and inhibit the release of 5-HT in the hippocampus in vitro (Balfour, 1980) and in vivo using microdialysis (Pei et al., 1989). Some of these studies demonstrated that this was due to an initial interaction at the ?-aminobutyric acid (GABAA) receptor complex since responses could be blocked by picrotoxin and flumazenil (Collinge et al., 1983; Pei et al., 1989). However, the relationship between the anxiolytic effects of benzodiazepines and their effects on 5-HT function is far from established. In one of the first studies of this kind, Wise et al. (1972) reported that the anxiolytic effects of benzodiazepines occurred at the same time as when the decreased 5-HT turnover occurred. Later studies have not, however, been able to replicate this finding (Lister and File 1983; McElroy et al., 1986). Instead, Lister and File (1983) suggest that the changes in 5-HT concentrations may be more closely related to the sedative effects of the drug. The knowledge that changes in 5-HT concentrations only occur following relatively large sedative doses of these drugs would also support this view (Pratt et al., 1979, 1985), although measurements of neurotransmitter concentrations are probably not very sensitive measures of neuronal events. Other biochemical studies have shown that systemic administration of diazepam and zopiclone reduced 5-HT synthesis in the hippocampus but not in other brain structures. This appeared to be a result of an action at the level of the hippocampus rather than at the origin of these 5-HT afferents in the median raphe. Focal injection of diazepam into the hippocampus reduced hippocampal 5-HT synthesis, an effect which was antagonised by flumazenil but not bicuculline, whereas infusion into the median raphe did not affect 5-HT synthesis (Nishikawa and Scatton, 1986). In contrast local injection of muscimol into the median raphe reduced 5-HT synthesis in the hippocampus, an effect that could be potentiated by benzodiazepines. The authors propose that benzodiazepines can exert inhibitory effects on hippocampal 5-HT neurons at two different neuroanatomical sites. Firstly, in the hippocampus at GABA receptor-independent benzodiazepine receptors located on, or close to, 5-HT nerve terminals and secondly, in the median raphe at benzodiazepine receptors coupled to GABA receptors. In addition, Nishikawa and Scatton (1986) suggest that the GABAergic control of serotonergic neurons is not tonically active in the median raphe since the raphe mediated effects on hippocampal 5-HT synthesis were only evident when GABA and benzodiazepine receptors were activated simultaneously. As a result of this they suggest that GABA-dependent benzodiazepine receptors in the median raphe do not play a major role in the overall effect of anxiolytic drugs on serotonergic transmission. However, others suggest that there is a tonic GABAergic control of 5-HT neurons in the median raphe (Forchetti and Meek, 1981) although this does not appear to be the case in the dorsal raphe (Gallager, 1978) The concept that GABA receptor-independent benzodiazepine receptors exist requires further investigation particularly since other groups have previously shown that the reduction of cerebral 5-HT turnover (albeit in the cortex) by benzodiazepines can be antagonised by picrotoxin (Collinge et al., 1983). A body of evidence is accumulating to suggest that benzodiazepines modify 5-HT release. In vitro studies showed that diazepam depressed potassium-evoked [3H]-5-HT release from hippocampal

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J.A. PRATT

synaptosomes (Balfour, 1980). Subsequently, Soubrie et al. (1983) found that in encephale isole cats systemic chlorodiazepoxide reduced the release of newly synthesised [3H]-5-HT in the basal ganglia, an effect which was blocked by flumazenil. Local application of chlordiazepoxide to the dorsal raphe resulted in a reduced release of 5-HT from the substantia nigra but not from other brain structures. Since direct application of this benzodiazepine to the basal ganglia or the dorsal hippocampus failed to change the local release of 5-HT from these areas, it seems reasonable to suggest that the action of chlordiazepoxide is more likely to involve the cell bodies rather than the nerve terminals of 5-HT neurons. More recently others using microdialysis have shown that systemic injections of flurazepam and diazepam reduce 5-HT release in the ventral hippocampus in conscious rats (Pei et al., 1989). However, in contrast to Soubrie's data, this effect was also observed following local perfusion of flurazepam into the hippocampus, suggesting an action in the vicinity of serotonergic terminals in the hippocampus. Taken together, these biochemical data clearly show that benzodiazepines modify 5-HT neuronal activity. A number of questions, however, remain to be resolved. Do benzodiazepines modulate 5-HT function at the level of 5-HT nerve cell bodies and/or terminals? Does this effect differ in different 5-HT neuronal pathways and how much does the degree of endogenous GABA tone in the distinct pathways contribute to the effects observed? Do GABA-independent benzodiazepine receptors exist and are these involved? Results from electrophysiological studies have suggested that benzodiazepines may modulate 5-HT neuronal activity through an action at the level of the cell bodies. Thus, systemic and iontophoretic administration of benzodiazepines potentiate the inhibitory effects of GABA and GABA agonists on the firing of serotonergic neurons in the dorsal raphe (Gallager, 1978; Trulson et al., 1982). It is proposed that the inhibitory actions of GABA on raphe neurons require the activity of a pathway from the habenula (Nishikawa and Scatton, 1985). Based on the electrophysiological data, to a certain extent on the aforementioned biochemical data and results from focal injection studies (see Section 4.1), it is tempting to propose that benzodiazepines exert their anxiolytic effect through inhibiting 5-HT raphe cell firing which in turn produces reduced 5-HT transmission in limbic structures. More direct experimental evidence for this arises from studies in which the 5-HT system has been manipulated in some way and the resulting behavioural effects of benzodiazepines in anxiety tests observed. In a conflict test Wise et al. (1972) reported that the release of behavioural suppression evoked after oxazepam administration is antagonised following the concurrent intraventricular administration of 5-HT. Subsequently, Tye et al. (1977) demonstrated that 5,7-DHT lesions of ascending serotonergic pathways mimicked the antipunishment effect of benzodiazepines and that chlordiazepoxide was unable to further release punished behaviour in the lesioned rats. Furthermore, lesions of the dorsal raphe by 5,7-DHT prevented the anxiolytic effects of chlordiazepoxide when administered into the raphe (Thiebot et al., 1982). Taken together, these findings support a reduction in 5-HT turnover as being a mechanism by which benzodiazepines exert their anxiolytic effects. However, other studies do not support this view. 5,7-DHT lesions of the dorsal raphe and substantia nigra do not prevent the ability of benzodiazepines to release punished responding when they are administered systemically (Thiebot et al., 1984b). The authors postulate that different neuronal systems are implicated in the release of behaviour induced by systemic versus centrally injected benzodiazepines. Moreover, they argue that one reason for the differences between their results and those of Tye and colleagues may be because the lesioned rats in the Tye et al. (1977) study had already reached their highest possible rate of response and so a response releasing effect of chlordiazepoxide could not be detected. Shephard et al. (1982) found that neither the 5-HT agonist 5-MeODMT, nor the 5-HT synthesis inhibitor PCPA could affect changes in punished responding evoked by chlordiazepoxide. They propose that benzodiazepines act distal to 5-HT synapses on a pathway mediating conflict responding. In summary, the role of 5-HT neuronal systems in anxiety remains controversial. Most of the behavioural tests employed rely on response suppression and this has led to the suggestion that 'anxiolytic effects' evoked in such tests may involve recruitment of 5-HT projections to the basal ganglia for the expression of motor behaviour. More recently, Soubrie (1988) has questioned whether the release of suppressed responding in conflict tests is synonymous with an anxiolytic effect. He postulates that it is attributable to a reduced ability to withold a response (reduced waiting ability or impulsivity).

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4. NEUROANATOMICAL SITES OF ACTION OF ANXIOLYTIC AND ANXIOGENIC D R U G S IN MODIFYING ANXIETY-RELATED BEHAVIOURS: FOCAL INJECTION STUDIES 4.1. BENZODIAZEPINERECEPTOR LIGANDS The diverse psychopharmacological profile of benzodiazepines is well documented. Nevertheless the various behavioural effects evoked by these drugs are all considered to be a result of an enhancement of cerebral GABAergic transmission. However, the neuroanatomical sites and pathways through which the behavioural effects are expressed remain poorly defined. 4.1.1. Neuroanatomical Locations of Receptors

The neuroanatomical locations of benzodiazepine receptors in brain have been investigated using receptor autoradiography. The receptors are extensively distributed throughout the CNS albeit with different densities in different structures (Marcel et al., 1986; Young and Kuhar, 1980). This would suggest that benzodiazepines are capable of modulating activity in many regions and circuits and this may underlie the multiplicity of behavioural effects evoked by these drugs. One approach employed to identify the possible neuroanatomical basis of action of the various behavioural effects of benzodiazepines is to examine the distribution of binding sites and deduce from regional heterogeneities the loci of benzodiazepine action. Hence, Young and Kuhar (1980) have hypothesised that receptors in the limbic system might mediate the anxiolytic effect, those in the cortex and limbic system might mediate the anticonvulsant effect, while receptors in the cerebellum, reticular formation and spinal cord might mediate the muscle-relaxant effect. Such an approach makes at least two assumptions. Firstly, it assumes previous knowledge of the functional role of different structures and secondly, it assumes that the degree of binding in a brain structure determines the importance of that region in the expression of the behavioural effect of a drug. This is a dangerous assumption, as it must be borne in mind that benzodiazepines can only modulate the efficiency of GABAergic transmission. Hence the effect of a benzodiazepine receptor ligand on a brain structure will depend not only on the density of benzodiazepine receptors but also upon the local degree of existing GABAergic tone. Results obtained from receptor autoradiography studies thus, do not directly indicate which structures are most functionally important in anxiolysis and which play a lesser role. 4.1.2. Focal Intracerebral Injections of Benzodiazepines

An alternative approach is to administer benzodiazepine receptor ligands into discrete brain structures and examine the subsequent anxiolytic or anxiogenic effects in animal tests of anxiety. However, with the knowledge that benzodiazepine receptors exist in many brain circuits and the fact that different neuronal systems may be involved in different aspects of anxiety, it is perhaps not surprising that anxiolytic effects can be evoked following focal injections into several different brain structures as illustrated in Table 2. There is a substantial body of work implicating the amygdala in the anticonflict affects of benzodiazepines although controversy exists over the relative importance of the various amygdaloid nuclei in mediating these effects. In one of the early studies Shibata et al. (1982) reported that benzodiazepines evoked an anticonflict effect in a Geller-Seifter type paradigm when the drugs were injected into the central but not into the basolateral and medial amygdala. These results tie in with the findings that lesions of the central amygdala evoke anticonflict effects in a Geller-Seifter type paradigm (Shibata et al., 1986) and mimic the effects of benzodiazepines in blocking conditioned fear, as measured in the potentiated startle paradigm (Hitchcock and Davis, 1986; see Section 2.3). Other studies have, however, implicated other amygdaloid nuclei as being important in the anticonflict effects of benzodiazepines. Thus, anticonflict effects are reported when benzodiazepines are injected into the lateral and/or basolateral nuclei (Scheel-Kruger and Petersen, 1982; Petersen et al., 1985; Thomas et al., 1985; Hodges et al., 1987) in both Geller-Seifter and water-lick conflict tests. In one study these effects were blocked by the benzodiazepine antagonist flumazenil (Hodges

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TABLE 2. Effects of Intracerebral Injections of Benzodiazepine Receptor Agonists in Behavioural Tests of

Anxiety

Authors

Structure

Drug

Test

Effect

Nagy et al., 1979

Amygdala

DZP

FSC

anticonflict

Hodges et al., 1987

AL/ABL

CDP MDZ

FSC

anticonflict anticonflict

Thomas et al., 1985

AL

CDP

FSC

anticonflict

Petersen et al., 1985

AL and ABL

MDZ

WSC

anticonflict

Scheel-Kruger and Petersen, 1982

AL and ABL AM and AC

MDZ, DZP

WSC

anticonflict no effect/mildly anticonflict

Shibata et al., 1982

AC AM and ABL

DZP, CDP and MDZ

WSC

anticonflict no effect

Shibata et al., 1989

ACE and ABL(ant)

LMZ, ZOP, DZP and FZP

WSC

anticonflict

Costall et al., 1989

Amygdala DR and MR

DZP

LDB

antiaversive

Thiebot et al., 1982

DR

CDP

FSC

anticonflict

Higgins et al., 1986

DR

FZP

SI and WSC

increased SI, not anticonflict

Kataoka et al., 1982

MB

DZP, CDP, and MDZ

WSC

anticonflict

Audi and Graeff, 1984

PAG

MDZ, CDP

Stimulated Aversion

reduced aversion

Yamashita et al., 1989a

MB AC, FC, dHC

ZOP

WSC

anticonflict no effect

Brain structure--Amygdaloid nuclei; AA, anterior; AB, basal; AC, central; ABL, basolateral; AL, lateral; AM, medial. Others--dHC, dorsal hippocampus; DR, dorsal raphe; MR, median raphe; FC, frontal cortex; MB, mammillary body; PAG, periaqueductal grey. Tests--FSC, food/shock conflict; WSC, water/shock conflict; LDB, light/dark box; SI, social interaction. Drugs---CDP, chlordiazepoxide; DZP, diazepam; MDZ, midazolam; FZP, flurazepam; ZOP, zopiclone; LMZ, lormetazepam.

et al., 1987). In contrast injections into the central or medial nucleus were minimally effective

(Scheel-Kruger and Petersen, 1982). More recently the Japanese group compared the effects of lesions of the central and of the basolateral nucleus in the water-lick conflict paradigm. In this study the results supported a role for the anterior part of the basolateral amygdala and the central amygdala but not the posterior basolateral amygdala or medial amygdala in conflict behaviour (Shibata et al., 1989). Benzodiazepines and the cyclopyrroline, zopiclone evoked anticonflict effects when administered into the anterior central amygdala which could be blocked by flumazenil. Clearly the reasons for these differences are difficult to explain. One possibility is that benzodiazepines could readily diffuse between these closely located nuclei. Another is that in some studies larger concentrations of benzodiazepines are required to evoke effects when injected into the central as compared to the basolateral site (Shibata et al., 1982; Scheel-Kruger and Petersen, 1982) raising the possibility that the drug may have diffused from the central to basolateral region. Some support for the basolateral region as being an important site for benzodiazepine action is that much higher densities of benzodiazepine receptors are found in the basolateral nucleus as compared to the central nucleus (Niehoff and Kuhar, 1983). Very few studies have investigated the effects of the intracerebral injection of anxiogenic benzodiazepine receptor ligands. However, Jones et al. (1986) reported that flCCM (methylcarboline-3-carboxylate) had an anxiogenic effect in the social interaction test when injected into the

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dorsal raphe. N-methyl-fl-carboline-3-carboxamide (FG 7142) and CGS 8216 (2-phenylpyrazolo4,5-c-quinolone-3(5H)-one) were shown to possess proconflict effects when injected into the amygdala (Hodges et al., 1987). These compounds also counteracted the anticonflict effects of chlordiazepoxide suggesting that the responses were mediated at the benzodiazepine receptor. Anticonflict effects of benzodiazepine agonists have also been reported following focal injection of these ligands into the mammillary body (see Table 2). In support of the mammillary body playing an important role in the actions of benzodiazepine receptor ligands, lesions of this structure attenuate the anxiolytic and anxiogenic effects of diazepam and FG 7142, respectively, in the elevated plus-maze test* (Laurie et al., 1990). Focal injections of benzodiazepines into the dorsal raphe produce less robust anxiolytic effects. Whilst Thiebot et al. (1982) demonstrated an anticonflict effect of chlordiazepoxide, Higgins et al. (1986) did not demonstrate an anticonflict effect of flurazepam but did show an anxiolytic effect in the social interaction test at large doses. Overall, these data suggest that certain amygdaloid nuclei and the mammillary body are important for the anxiolytic actions of benzodiazepines as determined from conflict tests. The role of the raphe nuclei is more controversial, particularly when evidence from lesioning studies is also taken into account (see Section 3.4). 4.2. 5-HT RECEPTORLIGANDS Despite the fact that the 5-HTIA agonist, buspirone is clinically effective as an anxiolytic its precise mechanism of action is unclear. Focal injections of buspirone, ipsapirone and 8-hydroxy-2(di-n-propylamino) tetralin (8-OH-DPAT) into the dorsal raphe produce anxiolytic effects in the social interaction and water-lick conflict test (Higgins et al., 1988). At larger doses these 5-HTmA agonists reduced 5-HT turnover in the frontal cortex, hippocampus and hypothalamus leading the authors to suggest that an action on 5-HT~A autoreceptors in the raphe decreased raphe cell firing and thereby reduced functional activity in the forebrain. In accord, focal injections of buspirone into the dorsal raphe were anxiolytic in the mouse light-dark box (Costall et al., 1988) although others claim the median raphe to be more important (Carli and Samanin, 1988). These findings do not however, exclude an action of these drugs on receptors distant from the nerve cell bodies. For example, Kostowski et al. (1989) reported an anxiolytic effect of buspirone in the plus-maze following injections into the dentate gyrus of the hippocampus, although the doses required were at least 10 times higher than those required to elicit effects in the dorsal raphe (Higgins et al., 1988). In contrast, injection of 8-OH-DPAT into the amygdala tended to produce an anxiogenic effect (Hodges et al., 1987). Interestingly, others report an anxiolytic effect of 8-OH-DPAT in the elevated plus-maze which is shifted to being anxiogenic following 5,7-DHT lesions of the dorsal raphe (Critchley and Handley, 1989). The 5-HT2 receptor antagonists, ritanserin and ketanserin, have been little explored with respect to their neuroanatomical basis of action. This is probably related to the fact that they are not always anxiolytic in animal tests of anxiety and that they show relatively weak activity with effects confined to a narrow dose range (Chopin and Briley, 1987). However, 5-HT2 receptors have been implicated in the antiaversive role of 5-HT in the dorsal PAG on the basis that focal injections of ketanserin into this region blocked the antiaversive effect of 5-HT (Schutz et al., 1985). More recent studies have led to the suggestion that functional interactions take place between several receptors in the in vivo control of PAG aversion with 5-HTjc receptors having a predominant role over the 5-HT 2 receptor subtype and with 5-HT 3 receptors having a minimal role (Jenck et al., 1990). In other tests of anxiety, such as the social interaction test, the proposed 5-HT~c receptor agonists 1-(3-chlorophenyl) piperazine (mCPP) and 1-(3-(trifluoromethly)phenyl) piperazine (TFMPP) display anxiogenic activity (Kennett et al., 1989). Results from focal injection studies suggest that this is due to an activation of 5-HT~c receptors in the hippocampus rather than the amygdala (Whitton and Curzon, 1990). It will be of interest to determine whether selective 5-HT~c antagonists are anxiolytic. *Laurie, D. J. and Pratt, J. A. Mammillary body lesions attenuate the anxiogenic action of FG 7142 in the elevated plus-maze. Pharmac. Biochem. Behav., submitted for publication.

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Handley (1991) has attempted to explain the bidirectional effects of certain 5-HT receptor ligands in different tests. She proposes that the behavioural inhibition system is not uniquely linked to anxiety but may be involved in other processing systems too. For example, a drug which is supposedly anxiolytic by enhancing behavioural inhibition, may be doing so by an increased readiness to make a response. Such a drug could in the same way display anxiogenic responses in models that measure response emission (Handley, 1991). There is as yet limited information on the neuroanatomical sites of action of 5-HT 3 antagonists in relation to their putative anxiolytic effects (see Costall et al., 1990). In the mouse light-dark box, injection of ondansetron and ICS 205-930 into the dorsal raphe nucleus and into the amygdala reduced the aversive response, whereas injection into the median raphe was less effective. In contrast, injections into the nucleus accumbens and striatum were ineffective, Interestingly, diazepam displayed a similar profile but injections into the median raphe produced similar antiaversive reponses as those occurring after dorsal raphe injections (Costall et al., 1989). The authors suggest that the ability of diazepam to attenuate the aversive response may be analogous to the effects of benzodiazepines in releasing behaviour suppressed by punishment following dorsal raphe injections in the rat (Thiebot et al., 1982) but a possible role of the median raphe is not discussed. Since both the dorsal and median raphe project to the amygdala it is possible that these pathways are critical to the behavioural response. The 5-HT 3 antagonists would also appear to act on the dorsal raphe-amygdala pathway, but the exact location of these receptors in both these structures is currently unknown. 5. FUNCTIONAL BRAIN MAPPING DURING ANXIETY AND RELATED BEHAVIOURS 5.1. THE INFLUENCEOF ANXIOGENIC DRUGS AND STRESSFUL STIMULI UPON LOCAL RATES OF CEREBRAL FUNCTIONAL ACTIVITY IN RODENTS

The preceding discussion has highlighted that both lesioning and focal injection studies can reveal individual structures involved in anxiety. However, neither approach permits an analysis of

Entorhinal cortex

FIG. 5. Diagrammatic representation of neuronal connections between limbic and functionally associated areas. Shaded areas indicate structures in which local cerebral glucose use was significantly increased by FG 7142 (3 and 10mg/kg) (see Pratt et al., 1988).

J.A. PRATT

FIG. 6. Autoradiograms prepared from coronal sections of rat brain depicting the effect of FG 7142 upon local cerebral glucose use. Note that FG 7142 (bottom section) markedly increases glucose use in the anterior thalamus and the cingulate cortex compared to control (top section) (see PraIt et al., 1988).

171

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173

the neuronal systems activated in a particular anxiety-provoking situation at the level of integrated neuronal networks. A different experimental strategy which can address this issue is the quantitative ~4C-2-deoxyglucose autoradiographic technique (Sokoloff et al., 1977). This technique permits the simultaneous assessment of local cerebral glucose use throughout the brain in the conscious animal without the necessity to choose structures for investigation prior to the experiments. Changes in glucose use following a particular pharmacological or physiological stimulus reflect changes in activity predominantly in nerve terminals and so the technique provides a complete neuroanatomical map of the structures affected as a consequence of receptor occupation (McCulloch, 1982). This technique can be particularly powerful when changes in local cerebral glucose use are correlated with a specific behavioural event. It is therefore possible to investigate the relationship between anxiety and alterations in local neuronal activity throughout the brain. Studies in our laboratory have shown that in anxiogenic doses the benzodiazepine receptor ligand FG 7142 evokes marked increases in glucose use in components of the Papez circuit (mammillary body, anterior thalamus and posterior cingulate cortex; increases of 32-50%) (see Figs 5 and 6). Increases occurred to a lesser degree in the septohippocampal formation (31-36%) and the basolateral amygdala (25%). In contrast, motor structures were minimally affected, although changes also occurred in structures associated with auditory and visual processing (Pratt et al., 1988). The latter may be related to the known ability of FG 7142 to increase arousal. This study (Pratt et al., 1988) has therefore demonstrated the simultaneous activation of limbic system structures by FG 7142 (see Fig. 5) separately implicated in anxiety by previous lesioning and focal injection studies. It also suggests a hierarchy of responsiveness between structures, with the Papez circuit playing the major role in this drug-induced anxiety. It will be of future interest to determine whether other anxiogenic stimuli recruit similar or different neuronal circuits to those recruited by FG 7142. Whilst there is a paucity of information on the neuronal systems activated during behavioural models of anxiety at the level of integrated neuronal networks, it is pertinent to note that changes in cerebral blood flow and metabolism have been investigated during exposure to stressful stimuli (Bryan, 1990). In this context stress is defined as a nonspecific response of an organism to a stimulus (stressor). The stimulus must be either an actual event that endangers the animal or one that is perceived as such. Hence Bryan (1990) includes hypotension, hypoglycaemia, hypoxia, conditioned fear and restraint as stressors. Perhaps most closely related to anxiety are the latter two stimuli. Following restraint of all four limbs cerebral glucose use was reduced in the hippocampus and anteroventral thalamus and increased in the lateral habenula (Soncrant et al., 1988). The habenula lies at the interface between limbic and motor structures and this may explain why changes occurred here. This procedure however only produced modest and generally insignificant reductions in cerebral blood flow (Ohata et al., 1981). In contrast, milder acute stress involving hindlimb restraint does not change energy metabolism (Soncrant et al., 1988; Bryan, 1990). It should be noted that changes in arterial pCO2 may alter cerebral blood flow. Indeed in the study of Ohata et al. (1981) it was estimated that changes in arterial pCO2 caused by hyperventilation which would tend to cause vasoconstriction may have offset any increases in blood flow caused by the stress. Conditioned fear produces more pronounced changes in cerebral blood flow and metabolism. The former was increased by 30-55% in limbic and auditory regions (Le Doux et al., 1983) whereas a 11-31% increase in cerebral glucose use occurred in limbic and a variety of other structures (Bryan and Lehman, 1988). In general, changes in flow to a specific brain area are a result of changes in the metabolic demand although flow and metabolism are not always tightly coupled. Bryan (1990) argues that stimulation of fl-adrenergic receptors may be a common feature of the changes in flow and metabolism evoked by stressful conditions. Certainly, propanolol can block increases in flow and metabolism during hypercapnia. However, plasma catecholamines alone do not increase cerebral flow and metabolism unless the blood-brain barrier is compromised as in the case of severe hypertension and osmotic insult (Mackenzie et al., 1976; Edvinsson et al., 1978). In the brain the locus coeruleus neurons provide the major noradrenergic supply and it is known that during stress there is an increased release of noradrenaline. Interestingly, stimulation of the locus coeruleus reduces cerebral bood flow (Goadsby and Duckworth, 1989). Taken together these results suggest that during stressful events the changes in cerebral blood flow and metabolism are unlikely JPT55/2--F

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J.A. PRATT

to be related to elevated plasma catecholamines (unless conditions become pathophysiogical) and the role of the locus coeruleus requires further investigation. It is more likely that the changes evoked are a combination of the animal evaluating the aversive stimulus together with it recruiting the neuronal systems required to express the emotional response. 5.2. POSITRONEMISSIONTOMOGRAPHYAPPLIEDTO INVESTIGATETHE NEUROANATOMICALBASISOF ANXIETY AND PANIC IN HUMANS In the clinic, positron emission tomography (PET) is being increasingly used to investigate structural and functional abnormalities in mental disorders and neurodegenerative disease states. The technique takes advantage of positron emission from radiopharmaceuticals to provide quantitative images of regional cerebral function. Glucose metabolism, oxygen utilization, Cerebral blood flow (CBF) and various neurotransmitter systems can be investigated by PET. Whilst current PET instrumentation requires further developments in terms of increased spatial resolution, the current resolution power of 5 mm and less has enabled important insights into the brain structures involved in the experience, evaluative and expressive components of anxiety (Reiman, 1988). PET also allows the opportunity to implicate regions of altered neuronal activity in the pathophysiology of anxiety-disorders. Reiman and colleagues 0984, 1986) have employed functional brain mapping studies to investigate panic disorder. It is well documented that sodium lactate infusions can precipitate a panic attack in patients suffering from panic disorder but rarely do so in normal controls. Reiman et al. (1984) in the first study of this type employed PET to study patients with panic disorder and normal controls prior to an infusion of lactate. Patients who subsequently went on to have a panic attack displayed an abnormal asymmetry of CBF (left < right) in a region of the parahippocampal gyrus, suggesting that there is a discrete brain abnormality in patients with panic disorder. Stewart et al. 0988) subsequently found that during lactate infusions, controls and patients who did not panic displayed marked increases in whole brain blood flow whereas patients who did panic displayed minimal changes in flow. Further investigations by Reiman et al. 0986) revealed additional abnormalities in the resting nonpanic state in patients who were vulnerable to lactate-induced panic. These were an abnormal asymmetry of blood volume and oxygen consumption in the parahippocampal gyrus, an abnormally high whole brain metabolism and abnormal susceptiblity to episodic hyperventilation. The authors suggest various possibilities for the parahippocampal gyrus abnormality: firstly, a relative or absolute increase in neuronal activity in the right parahippocampal region; secondly, a structural asymmetry and thirdly, a relative or absolute increase in blood-brain barrier permeability in the right hippocampal region. Based upon the first possibility, the theory of Gray (1987) and the knowledge that the area of the parahippocampal gyrus involved contains the major input and output to the hippocampal formation, Reiman et al. (1986) proposes that the abnormality reflects increased activity of local interneurons or an abnormal increase in the activity of projections to this region. These projection areas include the hippocampus, subiculum, entorhinal cortex, amygdala, raphe nuclei and locus coeruleus. If projections to the parahippocampal region are important in the evaluative aspects of this form of anxiety (i.e. vulnerability) then projections from the parahippocampal region to one or more of its outputs might be involved in the generation of a panic attack. One possibility is that the triggering event is via activation of noradrenergic projections to the hippocampus to generate an anxiety attack through the amygdala and its efferent connections (Reiman et al., 1986; Reiman, 1988). Recent investigations by the same group (Reiman, 1988) have shown that lactate-induced panic is associated with an increase in CBF in a variety of other structures (e.g. temporopolar cortex and lateral putamen). These have yet to be formulated into the neuroanatomical model of panic proposed by Reiman and colleagues. At present it is unclear whether abnormalities in the parahippocampal region represent a vulnerability to panic, a genetic predisposition or a component of anticipatory anxiety. A neuroanatomical model of panic which incorporates three components; acute panic attack, anticipatory anxiety and phobic avoidance has been proposed by Gorman et al. (1989). Panic attacks are proposed to originate in the locus coeruleus. Anticipatory anxiety is considered to be a result of altered activity in the limbic lobe as a result of activation of brain stem structures. Phobic

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avoidance is proposed to be a result of connections between the limbic lobe and the prefrontal cortex. The afferent pathways from the frontal cortex to brain stem structures would enable the latter to cause panic. Functional brain mapping studies have recently been extended to investigate anticipatory anxiety in healthy volunteers who were studied before, during and after anticipation of a painful electric shock (Reiman et al., 1989). Interestingly, during the anticipatory phase there was a raised blood flow in the temporal poles, the same regions implicated in the lactate-induced anxiety attacks in patients with panic disorder. Reiman et al. (1989) suggest that in normal volunteers the temporal poles could be involved in the evaluation process that characterises a situation with a sense of uncertainty and danger. In Reimans' view, panic disorder can be distinguished from normal forms of anxiety by the presence of a regional brain abnormality in the nonpanic state; an abnormality that could be involved in the initiation of the attack rather than be related to a state of anticipatory anxiety. Other groups have measured cerebral metabolism using PET in normal volunteers who displayed different scores of anxiety on the Spielbergers State-Trait Anxiety Inventory (Reivich et al., 1983, 1984). The high-anxiety group had higher metabolic rates in the right relative to the left hemisphere to a significantly greater extent than the low anxiety group. These data support the work of Reiman and colleagues (1989) and further suggest a greater right hemispheric involvement in high anxiety states. More recently, Gur et al. (1987) reported that normal volunteers who had low anxiety scores displayed a linear increase in cortical blood flow with anxiety. In contrast, subjects with higher anxiety scores displayed a linear decrease in CBF with increased anxiety, whereas there was a linear decrease in metabolic activity with increased anxiety. The authors suggest that the inverted U shaped relationship with the blood flow data and anxiety may be a physiological explanation of the Yerkes-Dodson behavioural law (Yerkes-Dodson, 1908) that stipulates an inverted U shaped relationship between anxiety and performance. It is also noted that the PET procedure for measuring metabolic rate is intrinsically more 'frightening' than the procedure of inhalation for measuring CBF. This may affect physiological values thereby questioning the ability to relate results from the two procedures. In summary, functional brain mapping studies have enhanced our understanding of the neuroanatomical basis of anxiety. However, it is clear that further work on patient populations suffering from different types of anxiety using instrumentation that can provide greater spatial resolution is required before a complete functional map of the structures involved in the different aspects of anxiety can be constructed with confidence.

6. SUMMARY AND CONCLUSIONS In summary, the available evidence suggests that anxiety is a heterogeneous disorder with each form having at least three components; the evaluation component that leads to the generation of anxiety, the subjective experience and the expression of anxiety. Each component is likely to recruit different neuroanatomical substrates which are presumably anatomically and functionally interconnected. Data from lesioning and electrical stimulation studies have revealed brain structures involved in the different experimental models of anxiety. However, in such studies it is difficult to discriminate between those structures involved in the subjective and those structures involved in the expressive components. Nevertheless, in two experimental models, the neuroanatomical circuitry recruited is reasonably well documented. In the fear-potentiated startle paradigm the amygdala and its connection with brain stem structures play a crucial role (see Fig 4). In contrast, in conflict models numerous limbic system structures are recruited. These include structures of the Papez circuit, septohippocampal formation, amygdala and brain stem structures such as the raphe nuclei (see Fig 2). The neuronal systems involved in ethologically based models of anxiety have been less intensively investigated. Current findings suggest an involvement of forebrain 5-HT projections from the raphe although further work is required to elucidate the contributions of other structures.

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Much of the work concerning the neuroanatomical sites of action of anxiolytic drugs has arisen from investigations of focal injections of benzodiazepines into discrete brain structures and observing the response in a behavioural test of anxiety. These drugs evoke anxiolytic responses in conflict tests following focal injections into a variety of brain structures. Perhaps this is not too surprising since benzodiazepine receptors are widely distributed in limbic system structures and are present in those structures (e.g. mammillary body and amygdala) that have been previously implicated in conflict behaviour from lesioning studies. Controversy still remains, however, over the role of the raphe nuclei in anticonflict responses to these drugs. The brain structures involved in the anxiolytic responses to benzodiazepines in ethologically based models of anxiety are less well documented; both the dorsal raphe and the mammillary body have been implicated. The raphe nuclei would appear to feature prominently in the anxiolytic actions of 5-HTIA agonists and 5-HT3 antagonists although few other structures have been investigated. Taken together, these data suggest that in order to determine the neuroanatomical basis of action of anxiolytic drugs it is important not only to take into account the distribution of receptors for such drugs but also to take into account the neuronal systems activated by the experimental model employed. Functional brain mapping studies can provide important information on the neuronal systems activated in a particular anxiety-provoking situation at the level of integrated neuronal networks. To date, results from animal studies have been limited to investigations of drug-induced anxiety but could be profitably employed to identify the neuronal systems recruited in the different aspects of anxiety in the different experimental models. PET studies in humans have revealed that in panic disorder there is focal brain abnormality in the parahippocampal gyrus. Interestingly, this same region displayed changes in blood flow in normal volunteers experiencing anticipatory anxiety. In the future, the application of functional brain mapping techniques together with studies concerned with the measurement of changes in neurotransmitter release in discrete brain structures in a particular anxiety-provoking situation, should provide new insights into our understanding of the functional neuroanatomical bases of anxiety disorders. Acknowledgements--I am grateful to the Wellcome Trust for supporting the work conducted in my laboratory.

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