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The Limbic System and Emotion in Relation to Acupuncture
Anthony Campbell
Patients receiving acupuncture sometimes manifest phenomena such as laughter or crying; varying degrees of relaxation or euphoria are quite common. Rarely, epileptic fits occur. Patients vary considerably in their responsiveness to acupuncture: some fail to respond at all while others (strong reactors) experience marked effects. It is widely recognised that fear of acupuncture generally precludes a therapeutic response, whereas belief in the efficacy of the treatment is irrelevant. All these phenomena must presumably have a neurophysiological explanation. This paper proposes that they may, at least in part, be caused by processes occurring in those brain structures that are classified as forming the limbic system. The paper briefly reviews the history and status of the limbic system idea, discusses how limbic system structures may contribute to the phenomena in question, and offers a number of predictions which would allow these hypotheses to be tested.
Key words
Acupuncture, Emotion, Laughter, Limbic system,
Seizures, Strong reactors.
Unexplained laughter or tears, though a little unusual, occur from time to time during acupuncture treatment and can be quite striking and prolonged. These reactions have usually been attributed to release of endogenous opioids, but there is not much direct evidence to support this idea, and one fact which counts against it is the observation that the response usually occurs very rapidly after the needles are inserted, even within seconds; too soon, one might think, for a humoral effect of that kind. Something that is seen more frequently than actual laughter or tears is euphoria. Some patients may report that they feel high, as if they have taken alcohol or other mood-altering drugs. This, again, is often attributed to release of endogenous opioids, but the same comments apply.
Marked euphoria is not particularly common, but minor versions of it, generally described as a sense of calm or relaxation, occur in probably 30 or 40 per cent of cases.
Another effect, which is fortunately much less common than those I have just described, is an epileptic fit. Fits generally happen in patients who are not known to be epileptic. When they occur in patients who were sitting at the time they are usually attributed to vasovagal syncope causing temporary cerebral anoxia, but this may not be the whole story since they can also occur in patients who are lying down. Also, it is well known that patients who are unwilling to have acupuncture or are afraid of it seldom respond well to treatment; this has been noted in the traditional literature for many centuries (on the other hand, belief in the efficacy of the treatment is unimportant).
All these things must, presumably, have a neurological explanation. They point to the occurrence of events in the central nervous system that have an effect on mood and behaviour, and what I should like to do is discuss some of the brain structures that may be involved. Much of the current theorising about acupuncture concentrates on the spinal cord and brain stem, with particular reference to pain (1). However, this does not tell us much about the subjective phenomena of euphoria and so forth, so I think the higher levels also need to be considered, particularly the limbic system.
Limbic system
There is a widespread idea that the limbic system is a part of the brain closely concerned with emotion, so it might reasonably be thought to have something to do with unexplained laughter and tears, as well as with fear and euphoria. Actually, the idea that the limbic system is concerned with emotion is at best a half-truth, but there certainly is a connection, which is probably relevant to acupuncture. But before getting down to this we need to establish exactly what it is we are talking about, because the limbic system is a rather slippery concept.
The limbic system is made up of the limbic lobe
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Corpus callosum
Anterior commissure
Cerebral cortex
Figure 1. Saggital section through the brain (3).

The limbic lobe is on the inner aspect of the cortex, It partially surrounds the corpus callosum and is made up of the cingulate (which means belt- like) gyrus and the parahippocampal gyrus. These are connected to each other by a narrow isthmus beneath the splenium (posterior part) of the corpus callosum. The hippocampus, which is in the floor of the temporal horn of the lateral ventricle, is also included in the limbic lobe. Other structures in the limbic system (Figure 2) are:
and certain additional structures. Anatomically, the main structures are on the inner or medial surface of the brain (Figure 1) and become visible only when it is split sagitally into two halves (2,3). Limbic means rim, as in the rim of a tennis racquet. One might wonder how the limbic lobe got that name, because in the human brain it is not all that conspicuous owing to the huge development of the cortex, but in other mammals, for example the rabbit, it forms a considerable part of the medial aspect of the hemisphere and really is a kind of rim, as the name implies. This was why Paul Broca coined the term in the 19th century. It was an anatomical, not a functional, description. Later this part of the brain was thought to be concerned with smell and was therefore called the rhinencephalon (meaning nose brain).
Dentate gyrus
  • Amygdaloid body (amygdala)
  • Hypothalamus (especially the mammillary
  • Septal area, in the basal forebrain
  • Thalamus (anterior and some other nuclei)
  • The amygdala This is a group of nuclei situated in the anterior part of the temporal lobe, lying between the anterior end of the temporal horn of the lateral ventricle and the ventral surface of the lentiform nucleus. Some of the nuclei are olfactory but most are involved in the limbic system.
    The hippocampus This forms an elevation in the floor of the temporal horn of the lateral ventricle. If you trace the structure outwards, towards the surface of the brain, you find that it is continuous with the parahippocampal gyrus at the inferiomedial surface of the temporal lobe; the hippocampus, in other words, is a cortical structure.
    To understand how this works, we need to take account of embryology and also the evolutionary history of the brain (5). What happens during its development is that the free margin of the developing brain (the pallium) becomes rolled upwards and then inwards to form the hippocampus. The part that is still at the surface, so to speak, is the parahippocampal gyrus. This folding process continues, now in the opposite direction, taking the free margin of the developing hippocampus outwards again, towards the medial surface of the temporal lobe. In this way the free margin of the hippocampus becomes doubled out and forms the dentate gyrus, which is separated from the parahippocampal gyrus by the hippocampal sulcus (dentate refers to the fact that its margin is serrated). As a result of this repeated

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    Cingulate gyrus
    Frontal lobe
    Olfactory bulb
    Spinal cord
    Mammillary body
    Figure 2. Diagram of the limbic system - anterolateral view (4).

    The name hippocampus means seahorse, but I think myself that it more closely resembles a scorpion without legs. The head of the scorpion lies close to the amygdala at the tip of the temporal lobe and the body extends posteriorly, ending below the splenium of the corpus callosum. The tail of the scorpion is represented by a band of efferent and afferent fibres which start as a ridge along its medial border called the fimbria. The left and right fimbriae continue as the crura of the fornix, which run from the hippocampus backwards, upwards, and then forwards beneath the corpus callosum. The tails of the two hippocampuses, i.e. the two fimbriae, join up for a time to form the body of the fornix before separating again to descend as the columns of the fornix and finally reach the mammillary bodies of the hypothalamus on each side.
    Histologically, the parahippocampal gyrus is 6-layered or almost so (i.e. neocortex) but the hippocampus is 3-layered (archicortex); there is a transition in structure at the bend called the subiculum. The pyramidal cell layer of the hippocampus is continuous with layer 5 (internal pyramidal) of the cortex. Certain of the
    hippocampal cells are thought to show an effect called long-term potentiation (LTP), which lasts for several days and is believed to be involved in the formation of new memories. The hippocampus varies in size in different animals and reaches its largest absolute and relative size in humans. This suggests that it has some important role in human consciousness.
    Pathology: The hippocampus is frequently damaged in disease (2). It is classified in three sectors, and the cells in the first of these (CA1) are particularly sensitive to oxygen deprivation. Temporary loss of oxygen supply is thought to be the cause of transient global amnesia. More prolonged loss, if bilateral, results in permanent failure to make new memories. Cells in CA1 are affected early in Alzheimer's disease. The limbic system has been implicated in schizophrenia: there is reduction in the size of the amygdala, hippocampus, and parahippocampal gyrus.
    Functional implications: These facts suggest that the hippocampus, and the limbic system generally, are closely connected with memory formation. So why do we have this idea that they are connected with emotion? To understand this, we have to see how the limbic system concept has developed (5).
    folding the mature hippocampus comes to consist of two interlocking sections of tubes. Hence we can speak of the hippocampal formation, consisting of the hippocampus, the dentate gyrus, and most of the parahippocampal gyrus.

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    A brief historical digression
    At the end of the 19th century the philosopher and psychologist William James put forward a theory of emotion which seemed at first glance paradoxical. Common sense says that, when you see a bear in the wood (James's example), you feel frightened: your stomach churns, you start to sweat, and you run away. This implies that the emotion of fear causes the physical effects. James, however, said that seeing the bear directly causes the physical effects of sweating, churning stomach, and running away, and it is the awareness of these physical events that constitutes the feeling of fear. In other words, in James's view the emotion is caused by the physiological changes, not the other way about.
    In the 1920s Walter Cannon and Philip Bard put forward a somewhat similar theory. They suggested that seeing the bear causes changes in the thalamus; these in turn are transmitted to the hypothalamus and thence to the muscles and organs, and it is the events in your brain that are perceived as fear. This is the famous fright and flight reaction. In other words, they moved the centre of interest from the periphery to the brain, and especially the hypothalamus. Cannon disagreed with James's idea, but in fact his own theory is not all that different; it just focuses on different physiological phenomena. There was, however, apparently a difficulty with this theory: the cortex was thought not to be directly connected to the hypothalamus, so how could changes in the hypothalamus reach consciousness? (We now know that the hypothalamus is in fact widely connected with almost all areas of the cortex.)
    Then in 1937 James Papez put forward a new theory which was destined to become very influential. He went back to Broca's notion and suggested that the limbic lobe was responsible for emotion. Information was supposed to flow in a circuit from the hypothalamus to the medial cortex and back again. The sequence is as follows: parahippocampal gyrus (entorhinal area) - hippocampus - mammillary bodies (hypothalamus) - thalamus (anterior nuclei) - cingulate gyrus - parahippocampal gyrus. This is the famous Papez circuit. In it, the cingulate gyrus has a central role; it is supposed to be the emotional cortex and analogous to the sensory cortex in the parietal lobe. The hippocampus is also concerned with emotion. This was a mistake, but nevertheless the theory was a brilliant anticipation of later anatomical discoveries.
    Paul MacLean took up the Papez theory in 1949 and elaborated it. He suggested that the hippocampus was what he called an emotional
    keyboard (a bit like the Organ of Corti?). He continued to elaborate this idea as the years went by and in 1970 he introduced the notion of the triune brain, made up as follows:
    i. Reptilian brain = brainstem reticular system and basal ganglia
    ii. Old mammalian brain = limbic system
    iii. New mammalian brain = neocortex Emotional and psychological disorders were supposed to be due to faulty communication between the old and new parts of the brain. This theory was popularised by Arthur Koestler, who used it to diagnose what was amiss with modern society, and reverberations of this notion continue to be felt today in much popular theorising.
    So where does all this leave the limbic system today? The limbic system idea is now generally accepted by brain scientists. Textbooks of neuroanatomy contain a chapter on it and lay dictionaries have an entry for it. Nevertheless, it has important defects.
    On the merit side, it sets emotion in an evolutionary context, and it may explain some of the psychiatric symptoms (dissociation of intellect and emotion) that MacLean claimed it did. But the distinction between old and new cortex is no longer accepted: for this reason some authorities now prefer to avoid terms like neocortex and archicortex and instead speak of isocortex and allocortex. Moreover, damage to the hippocampus and mammillary bodies has little effect on emotion but a great deal on memory, contrary to MacLean's prediction. All three structural and functional levels postulated by MacLean are found in reptiles and so- called lower mammals as well as in phylogenetically newer mammals. In any case, mammals cannot be plausibly divided into primitive and advanced groups. It can hardly be supposed that a brain system that has existed in its basic form for thousands of millions of years is not highly adaptive for survival and further evolution.
    So why has the limbic system concept survived so long? Partly because it is a convenient shorthand to name the areas between the hypothalamus and neocortex, and partly because these structures, especially the amygdala, do have something to do with emotion; the theory is not completely wrong. But to call the limbic system the emotional centre of the brain is certainly misleading (6,7).
    The limbic system and acupuncture
    I would like to suggest that acupuncture is capable of modifying the activity of the limbic system so as to give rise to the various phenomena I mentioned in the introduction. But first: is it plausible to suggest
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    that acupuncture could actually modify brain function in this way? I think it is, if we remember that much of the limbic system (anterior cingulate and hippocampus) is cortical or derived from cortex. Cortical representation is a lot more plastic than was realised a few years ago, and what is interesting in the present context is that this seems to be particularly true of painful stimulation.
    For example: In a recent study (8), healthy volunteers were given tactile stimulation to the right side of the lower lip for a few minutes. Then they were given painful electrical stimulation to the median nerve on the same side. When the lip stimulation was repeated, some of them felt this both at the lip and at the site in the wrist where the painful stimulus had been applied; one of these subjects actually felt that her fist was clenched, which is something that patients with a phantom limb sometimes report. This study thus suggests that even brief peripheral painful stimulation can temporarily alter sensory cortical mapping. I therefore find it quite plausible to suppose that it can also modulate activity in the anterior cingulate cortex and other limbic system structures.
    Although it is now known that the hippocampus is concerned with memory, the amygdala is indeed involved in emotion, especially fear (5).
    i. If both temporal lobes are removed from animals they become abnormally tame and are also hypersexual. They put all sorts of things in their mouths because they are unable to decide, from sight, if things are suitable to eat (Kluver-Bucy syndrome).
    ii. In humans, stimulation of the amygdala produces feelings of fear or anger, which suggests that activity in the amygdala causes the autonomic and somatic accompaniments of these emotions.
    iii. The amygdala (and other parts of the nervous system) have many post-synaptic receptors for which gamma-amino butyric acid (GABA) is an inhibitory neurotransmitter, and diazepam and other anxiolytics mimic the action of GABA at these sites.
    iv. Fear conditioning in animals (rather similar to Pavlovian conditioning but using innate fear- producing responses) results in very long-lasting effects which, unlike the responses produced by Pavlovian techniques, are probably permanent and are mediated by the amygdala.
    v. Seizures originating in the amygdala are associated with emotions, especially fear.
    These facts about the amygdala suggest the following hypotheses:
    i. Part of the calming effect of acupuncture may be due to release of GABA in the amygdala and other limbic system areas.
    ii. The reason why acupuncture does not work in patients who are afraid of it may be that their amygdalas are primed by early experiences of pain associated with needles so that they always react badly in similar situations (a form of fear conditioning). Belief, on the other hand, is a cortical phenomenon and does not affect this pathway directly.
    And these predictions can therefore be made: i. Imaging studies should show increased
    activation of the temporal lobes in strong reactors. ii. Animal studies should show that acupuncture causes release of GABA in the
    Strong reactors
    Why is it that some people react very strongly to acupuncture while others do not seem to react at all? Johnson and colleagues (9) have found that patients differ considerably in their response to transcutaneous electrical nerve stimulation (TENS). Each patient tends to have a preferred frequency and pattern of stimulation which is constant for the individual, so these authors suggest that lack of response to TENS may be linked to a low central nervous system responsiveness to sensory stimuli in general. It seems plausible that the same is true of acupuncture sensitivity. Strong reactors would then be people whose central nervous system, including the limbic system, is particularly sensitive to sensory stimulation. In the case of the limbic system there are nociceptive receptors in the cingulate cortex that have large receptive fields that may encompass the whole body (10). Strong reactors may therefore be people with many such receptors.
    Concerning euphoria, we may get a clue from experiments in which Ketter and colleagues used intravenous injection of procaine to modify the activity of the limbic system and related structures in normal subjects (11). Some of these people experienced euphoria which was related to reduced activity in the left amygdala. Visual and auditory hallucinations also occurred. Regional cerebral blood flow increased in anterior paralimbic structures, particularly areas related to the amygdala. The authors remark that there are also reports of euphoria being caused by stimulation of the anterior cingulate cortex.
    This suggests that strong reactors may be people in whom these structures are more easily influenced by sensory stimulation, either to depress the
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    amygdala or to stimulate the anterior cingulate. The following predictions about strong reactors, which relate to Mann's impression that they are artistic, etc. (12), can therefore be made:
    i. Strong reactors should show an increased incidence of a history of out-of-body experiences and hallucinations.
    ii. They may be more inclined to religious belief.
    Although rarely, some patients have had fits during acupuncture (13). All the structures we have been considering are frequently implicated in epilepsy, especially epilepsy with strange subjective phenomena, and not necessarily accompanied by loss of consciousness. This is called complex partial epilepsy, or temporal lobe epilepsy. Temporal lobe epilepsy can cause difficulties in speaking, and I have noticed that strong reactors occasionally seem to find some problem with this while being needled.
    There are similarities between temporal lobe epilepsy and some kinds of migraine aura, and the fact that acupuncture seems to help patients who suffer from migraine with aura may be connected with this. Moreover, the authors of the procaine study say that patients with anxiety disorders report an increased incidence of spontaneous paroxysmal and emotional phenomena similar to those of patients with complex partial epilepsy. This may be due to limbic dysfunction (12).
    The implication is that epilepsy occurring during acupuncture may at times be due to activation of the temporal lobes via their connections with the amygdala and hippocampus. Although frank fits are unusual, some of the phenomena that acupuncture patients report may be very minor versions of the same process. For instance, one 30-year-old epileptic woman experienced a warm sensation in both feet ascending through the legs (2). Similar sensations are described from time to time by acupuncture patients and are ascribed by traditionalists to the flow of Qi. It can be predicted that:
    i. Epilepsy should be more likely to occur in strong reactors.
    ii. Strong reactors should show EEG changes in their temporal lobes during acupuncture to a greater extent than other patients, even without suffering fits.
    Laughter and tears
    Not much is known about how laughter is produced by the brain. Papez and later writers have claimed that it is a motor programme organised by the limbic system and brain stem. Arroyo and colleagues have
    described three patients with seizures who shed light on this question (14). In one, the onset of seizures was in the left anterior cingulate region. This patient did not feel amused although she was laughing. The other two did not laugh during seizures but laughter was elicited by electrical stimulation of the parahippocampal gyrus and neighbouring cortex, and both of these actually felt amused during the stimulated laughter. These observations suggest that laughter can be produced by activation of parts of the limbic system, though there are differences according to the exact site of stimulation.
    i. Laughter without subjective amusement can be produced by seizures starting in the anterior cingulate region.
    ii. Electrical stimulation of the basal temporal lobe can cause laughing accompanied by a sense of amusement.
    One possibility, therefore, is that acupuncture simply activates those parts of the limbic system that are involved in the production of laughter or tears. Presumably it does this in a relatively normal way, since patients generally do feel amused or euphoric when this occurs. However, there is also another possibility, based on a suggestion by Ramachandran (15). Patients with a condition called pain asymbolia feel pain but do not experience the emotional accompaniment. Moreover, some patients with this disorder giggle when pricked. This, of course, is exactly what happens in acupuncture. So it is tempting to see a clue to why acupuncture causes laughter here, but there is a problem: pain asymbolia is pathological; it may be due to damage to the insular cortex, which receives pain input and projects to the anterior cingulate cortex, whereas acupuncture-related laughter usually occurs in the presence of an intact nervous system. However, it might be supposed that acupuncture can somehow temporarily disconnect the insular cortex from the anterior cingulate, to produce a kind of temporary pain asymbolia.
    There is yet another idea, related to the reason we laugh when we are tickled, again based on a hypothesis put forward by Ramachandran. He thinks that laughter generally occurs when we perceive an incongruity; this is the basis of many jokes and also the reason we may laugh when a pompous man slips on a banana skin. Ramachandran suggests that when we are tickled we perceive an incongruity between an apparent physical threat and a context of play; this makes us laugh. This theory explains why one cannot easily tickle oneself: one knows in advance that there is no threat involved.
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    Acupuncture is also a kind of physical threat, in that (relatively minor) pain is inflicted, but in a therapeutic context. Also, one may expect the needle to be painful and then be pleasantly surprised when it is not as bad as expected. There may thus be a functional dissociation between the insula and the anterior cingulate cortex. The insular cortex registers the needle prick as a threat, but the cingulate cortex does not agree, hence there is a perceived incongruity resulting in laughter. On this hypothesis acupuncture is an equivalent to tickling; certain predictions follow, which might be confirmed or otherwise quite simply by questionnaire.
    i. Patients who laugh when having acupuncture should find the procedure pain-free or nearly so (because severe pain would be likely to over-ride the dissociation).
    ii. These patients are also likely to be especially ticklish.
    iii. Part of the pain relief that acupuncture can produce may be due to dissociation between the sensation of pain and the emotional distress that it normally causes; patients may continue to feel pain but be less bothered by it.
    One final speculation: I know of no particular effect of acupuncture on memory, but in view of the importance of the hippocampus in memory formation and the fact that this structure is damaged early in Alzheimer's disease, one might predict that patients suffering from this disorder would be unlikely to respond well to acupuncture.
    Some acupuncture effects may be due to events in the limbic system; certain predictions follow from this hypothesis and should allow it to be tested. We need to know more about the personality and physiological characteristics of strong reactors; at present this is at the level of anecdote. Verification of these suggestions may take some time to carry out, but I believe that if we are to arrive at a reasonably comprehensive explanation of how acupuncture works we will need to take into account the role of the limbic system. In the meantime, if one reflects on what is known about this very complex system, it does shed light on some of the more curious phenomena which are encountered from time to time while needling patients.
    I should like to express my warm thanks to Professor John
    Thompson, who read an early draft of this paper and encouraged me to continue with it; he also very kindly provided some valuable references and reprints of his papers. I need hardly say that he is not responsible for any errors I may have stumbled into. I am also grateful to my son Robert, a neuroscience student, who obtained some essential reference material for me.
    Anthony Campbell MRCP FFHom 8 Oak Way, Southgate, London N14 5NN (UK) Email:
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    3. Beatty J. Principles of behavioral neuroscience. Brown and Benchmark; 1995.
    4. Bloom, Laserson. Brain, mind and behavior. USA: WH Freeman; 1988.
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    Henningsen H. Phantom sensations following acute pain.
    Pain 1998; 77: 209-13. 9. Johnson MI, Ashton CH, Thompson JW. The consistency of
    pulse frequencies and pulse patterns of transcutaneous nerve stimulation (TENS) used by chronic pain patients. Pain 1991; 44: 231-4.
    10. Treede RD, Kenshalo DR, Gracely RH, Jones AKP. The cortical representation of pain. Pain 1999; 79: 105-11. 11. Ketter TA, Andreason PJ, George MS, Lee C, Parekh PI, Willis
    MW, Herscovitch P, et al. Anterior paralimbic mediation of procaine-induced emotional and psychosensory experiences. Archives of General Psychiatry 1996; 53: 59-69.
    12.Mann F. Reinventing acupuncture: a new concept of ancient medicine. Oxford: Butterworth Heinemann; 1992.
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    1993; 116: 757-80. 15. Ramachandran VS, Blakeslee S. Phantoms in the brain.
    London: Fourth Estate; 1998.

    Musculoskeletal Disease
    by John Reynolds
    The course will be held on April 2nd 2000 in York and will be repeated on November 4th 2000 in Enfield.
    This one day course for medical practitioners covers the theory and practice of musculoskeletal treatment, both Traditional and Western: Trigger points, Meridian and TCM points, Periosteal pecking, Gunn’s Intramuscular Stimulation techniques, Auricular acupuncture and Myofascial syndromes. It is expected that doctors attending will have a basic knowledge of acupuncture, having attended an introductory course previously. Approved for CME and PGEA.
    Details from: B.M.A.S. Administrator Newton House, Newton Lane, Lower Whitley, Warrington, Cheshire. WA4 4JA Telephone: 01925 730727 Fax: 01925 730492 Email: Web:
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    The limbic system and emotion in relation to acupuncture
    Anthony Campbell
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    Cerebellum (2011) 10:61–69 DOI 10.1007/s12311-010-0228-z
    Periaqueductal Grey Stimulation Induced Panic-Like Behaviour Is Accompanied by Deactivation of the Deep Cerebellar Nuclei
    Véronique M. P. Moers-Hornikx & Johan S. H. Vles & Lee Wei Lim & Mustafa Ayyildiz & Sűleyman Kaplan & Antonio W. D. Gavilanes & Govert Hoogland & Harry W. M. Steinbusch & Yasin Temel
    Published online: 16 November 2010
    # The Author(s) 2010. This article is published with open access at
    Abstract Until recently, the cerebellum was primarily considered to be a structure involved in motor behaviour. New anatomical and clinical evidence has shown that the cerebellum is also involved in higher cognitive functions and non-motor behavioural changes. Functional imaging in patients with anxiety disorders and in cholecystokinin tetrapeptide-induced panic-attacks shows activation changes in the cerebellum. Deep brain stimulation of the dorsolateral periaqueductal grey (dlPAG) and the ventro- medial hypothalamus (VMH) in rats has been shown to induce escape behaviour, which mimics a panic attack in humans. We used this animal model to study the neuronal activation in the deep cerebellar nuclei (DCbN) using c-Fos immunohistochemistry. c-Fos expression in the DCbN decreased significantly after inducing escape behaviour by stimulation of the dlPAG and the VMH, indicating that the DCbN were deactivated. This study demonstrates that the
    V. M. P. Moers-Hornikx : L. W. Lim : G. Hoogland : H. W. M. Steinbusch : Y. Temel Department of Neuroscience, Maastricht University, P.O. Box 616, 6200 MD Maastricht, The Netherlands
    V. M. P. Moers-Hornikx (*) : J. S. H. Vles Department of Neurology, Maastricht University Medical Centre, P.O. Box 5800, 6202 AZ Maastricht, The Netherlands e-mail:
    L. W. Lim : G. Hoogland : Y. Temel Department of Neurosurgery, Maastricht University Medical Centre, P.O. Box 5800, 6202 AZ Maastricht, The Netherlands
    S. Kaplan Department of Histology & Embryology, Medical School of Ondokuz Mayis University, Samsun, Turkey
    DCbN are directly or indirectly involved in panic attacks. We suggest that the cerebellum plays a role in the selection of relevant information, and that deactivation of the cerebellar nuclei is required to allow inappropriate behaviour to occur, such as panic attacks.
    Keywords Cerebellum . Deep brain stimulation . Periaqueductal grey . Ventromedial hypothalamus . Escape behaviour
    CCAS Cerebellar cognitive affective syndrome OCD Obsessive-compulsive disorder CCK-4 Cholecystokinin tetrapeptide PAG Periaqueductal grey
    DBS Deep brain stimulation dlPAG Dorsolateral periaqueductal grey
    M. Ayyildiz Department of Physiology, Medical School of Ondokuz Mayis University, Samsun, Turkey
    V. M. P. Moers-Hornikx : L. W. Lim : G. Hoogland : H. W. M. Steinbusch : Y. Temel European Graduate School of Neuroscience (EURON), Maastricht, The Netherlands
    A. W. D. Gavilanes Department of Pediatrics-Neonatology, Maastricht University Medical Centre, P.O. Box 5800, 6202 AZ Maastricht, The Netherlands
    Cerebellum (2011) 10:61–69
    VMH Ventromedial hypothalamus DCbN Deep cerebellar nuclei OF Open field FN Fastigial nucleus of the cerebellum DN Dentate nucleus of the cerebellum IN Interposite nucleus of the cerebellum c-Fos-ir c-Fos immunoreactive
    PC Purkinje cell
    The cerebellum has long been considered to be predominantly involved in motor coordination and control [1, 2]. The last two decades, however, it has become increasingly clear that the cerebellum is also involved in behaviour and cognition [3, 4]. Schmahmann and Sherman described the cerebellar cognitive affective syndrome consisting of impairment of executive functions, difficulties with spatial cognition, changes of personality including disinhibited or inappropri- ate behaviour and language deficits [5]. Recent neuroana- tomical and functional imaging research has pointed towards a role for the cerebellum in neuropsychiatric diseases such as autism, schizophrenia and mood and anxiety disorders [6– 11]. With respect to anxiety disorders, Levinson described the existence of cerebellar-vestibular dysfunction in 94% of patients with various types of anxiety disorders [12]. De Bellis and Kuchibhatla showed that children and adolescents with post-traumatic stress disorder related to maltreatment have significantly smaller cerebelli than paediatric subjects with generalized anxiety disorder and healthy subjects [13]. Several groups have shown structural and functional differ- ences in the cerebellum of patients with anxiety disorders compared to control subjects [14–22]. In patients with obsessive-compulsive disorder (OCD), a disorder character- ised by anxiety, an increase in grey matter was found in the anterior cerebellum [22]. Using functional imaging, Busatto et al. found an increased activation in the superior cerebellum of OCD patients compared to control subjects [14]. Another group showed that after treatment, OCD patients showed a decreased activation in the cerebellum compared to pretreatment scans [15]. In contrast, Nabeyama et al. found decreased activation in untreated OCD patients compared to control subjects, which increased after successful treatment of these patients [16]. In patients with panic disorder, an increased activation of the cerebellum was found when compared to control patients [20]; this activation decreased again after successful treatment of these patients [21]. In healthy individuals in whom panic attacks were induced by cholecystokinin tetrapeptide (CCK-4), an increased activation of the cerebellum was found, especially in the vermis [23–26]. However, the significance of these findings is not yet clear.
    Panic disorder is one of the most frequently encountered anxiety disorders [27]. Panic attacks consist of an acute, sometimes unexpected onset of intense fear, accompanied by a variety of physical symptoms [27]. Treatment usually includes behavioural and pharmacological thera- pies, either alone or in combination. It is thought that panic attacks may be the result of an abnormally sensitive fear network [28]. The periaqueductal grey (PAG) is known to be part of this fear network which further consists of the central nucleus of the amygdala, ventro- medial hypothalamus (VMH), hippocampus and other brainstem regions [28]. Deep brain stimulation (DBS) in rats of the dorsolateral periaqueductal grey (dlPAG) and one of its target structures, the VMH has been shown to elicit a typical behaviour consisting of vigorous running and jumping [29]. This behaviour, also known as escape behaviour, is accompanied by strong emotional and autonomic activation, and thereby mimics panic attacks in humans [29, 30].
    In the present study, we used DBS of the dlPAG and the VMH as a model of panic attack to evaluate the involvement of the deep cerebellar nuclei (DCbN). In a previous study, we have found that increased impulsivity in rats was accompanied by a deactivation of the DCbN [4]. These findings suggested that the cerebellum plays a role in regulating appropriate behaviour. Based on their findings in a patient with pathological laughter and crying, Parvizi et al. hypothesized that the cerebellum adjusts behavioural responses to the correct situational context of a stimulus, and that when the cerebellum operates on the basis of incorrect information, this could lead to inappropriate or even chaotic behaviour [31]. We hypothesized that deacti- vation of the DCbN was needed for pathological behaviour to occur. In line with this, here we predicted to find again a deactivation of the DCbN in rats with panic-like behaviour. Panic attacks consist of an inadequate response to a stimulus. Possibly the cerebellum also plays a role in regulation of the response to these stimuli by selection of relevant information. Deactivation of the DCbN may then be linked to an inadequate response to a certain stimulus by disabling this selection process. To analyze activation patterns, we have used c-Fos immunohisto- chemistry. We focused on the DCbN as these are the output structures of the cerebellum. c-Fos was chosen as it is an immediate early gene which is considered to reflect neuronal activation [32, 33]. The level of c-Fos protein is maximal about 2 h after administration of a stimulus and disappears again after 4 to 8 h even if the stimulus is continued [32]. Therefore, the c-Fos expres- sion found 2 h after a specific stimulus in controlled conditions can be considered to result directly from the stimulus, and this expression can be used to identify brain areas influenced by this stimulus [33].
    Cerebellum (2011) 10:61–69
    Materials and Methods
    current amplitudes necessary to evoke escape behaviour, all rats had a period of 2 weeks without stimulation before the final stimulation session in the open field (OF) arena was performed. Control rats were handled and placed in the OF similarly.
    Behavioural Evaluation
    Rats were evaluated in an OF test. For more details of this test, please see Lim et al. [29]. In summary, rats were placed in the arena and were connected with an external stimulator through externalized leads. The stimulation started approximately 1 min after the rat was placed in the OF arena, using the current amplitudes previously eliciting escape behaviour. The behaviour of the rats was videotaped (Ethovision, Noldus Information Technology, Wageningen, the Netherlands).
    Histological Processing
    Two hours after the final stimulation session, rats were deeply anesthetized and perfused transcardially with Tyrode (0.1 M), followed by a fixative containing 4% paraformal- dehyde, 15% picric acid and 0.05% gluteraldehyde in 0.1 M phosphate buffer (pH 7.6). The brains were removed and postfixed for 2 h followed by immersion in 20% sucrose at 4°C until saturated. Brain tissue was then quickly frozen with CO2 and stored at −80°C. The cerebellum was cut serially into 10-μm sections, which were collected on gelatine-coated glasses. We used a previously published protocol for c-Fos immunohistochemistry [4]. In summary, this staining was carried out by incubating sections two nights with a polyclonal rabbit anti-c-Fos antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA).This was followed by overnight incubation with the secondary antibody (biotinylated donkey anti-rabbit, Jackson Immu- noresearch Laboratories Inc., Westgrove, PA, USA). Subsequently, the sections were incubated with an avidin–biotin–peroxidase complex (Elite ABC-kit, Vectastatin; Burlingame, CA, USA) for 2 h. To visualize the immune complex of horseradish peroxide reaction product, sections were incubated with 3,3-diaminobenzidine tetrahydrochloride (DAB)/nickel chloride (NiCl2) solution for 10 min. After dehydrating, all sections were cover-slipped with Pertex (HistolabProducts ab, Goteborg, Sweden). Additionally, for each animal, the area of the dlPAG and VMH was cut into 30-μm sections and stained with standard haematoxylin– eosin (Merck, Darmstadt, Germany) staining to locate the electrode tips.
    Stereological Quantification of c-Fos Immunoreactive Cells
    Stereological analysis was performed with a stereology workstation (CAST-GRID-Computer Assisted Stereological
    The animals used were male 12 weeks old, bred and housed at the Central Animal Facility of the Maastricht University, the Netherlands), with an average body weight of 300–350 g at the time of surgery. Rats were housed individually in standard cages with sawdust bedding in an air-conditioned room under a 12/12-h reversed light/dark cycle. Food, standard laboratory chow (Hopefarms, Woerden, the Netherlands) and water were available ad libitum. All experiments were approved by the Animal Experiments and Ethics Committee of Maastricht University.
    Surgical Procedure
    Rats were randomly assigned to one of the following three experimental groups: control (no surgery), dlPAG DBS or VMH DBS. A detailed description of the surgical procedure was reported previously [29, 34]. In brief, animals were anaesthetized throughout the entire procedure using a combination of ketamine (90 mg/kg) and xylazine (10 mg/kg) injected subcutaneously. Rats were placed in a stereotactic apparatus (Stoelting, Wood Dale, IL, USA; model 51653). After making a burr hole, rats received implantation of a unilateral electrode at the level of the right dlPAG (coordinates from Bregma, AP=−7.6 mm, ML= 0.7 mm and V = −4.8 mm; approached with a coronal angle of 10°) and the right VMH (coordinates from Bregma, AP=−2.5 mm, ML=0.5 mm and V=−9.5 mm). A construction of one electrode with an inner wire of a platinum–irridium combination (Technomed, Beek, the Netherlands), with a tip diameter of 50 μm and a shaft diameter of 250 μm, was implanted in this experiment [35]. The electrodes were fixed in position using dental cement.
    Deep Brain Stimulation
    After a recovery period of about 1 week, all animals underwent a first stimulation session to determine the escape threshold. The stimulation amplitudes were gradually increased until escape behaviour was observed. At each step, stimulation duration was 15 s followed by a period without stimulation of 45 s. The stimulation frequency was set at 50 Hz and pulse width at 0.1 ms based on previous experiments [29]. A World Precision Instruments digital stimulator (DS8000, WPI, Berlin, Germany) and a stimulus isolator (DLS100, WPI, Berlin, Germany) were used to deliver the stimuli. Real-time verification of the parameters applied during stimulation was obtained using a digital oscilloscope (Agilent 54622D oscilloscope, Agilent Tech- nologies, Amstelveen, the Netherlands). After confirming the
    albino Wistar rats
    (n = 15,
    Cerebellum (2011) 10:61–69
    Toolbox-Olympus, Denmark). After exactly tracing bound- aries of the fastigial nucleus (FN) and dentate nucleus of the cerebellum (DN) on microscopic video images displayed on a monitor, numbers of c-Fos immunoreactive (c-Fos-ir) cells in both nuclei were evaluated with the modified Abercrombie cell counting method [36]. All neurons of which the cell bodies gave a positive reaction within an unbiased counting frame distributed in a systematic-random fashion throughout the delineated regions were counted. Estimated numbers of neurons were calculated from the numbers of counted neurons and the corresponding sam- pling probability. The numerical density (NV) of c-Fos-ir cells in the nuclei was estimated using the formula NV= (NA/T) (T/(T+D)) [36]. In this formula, NA, T and D are the positive cell number in the unbiased counting frame, the section thickness and the mean particle diameter, respec- tively. To make an acceptable biological comment on any subject, one should estimate total number of cells rather than numerical density of cells in the interested region. For this reason, we estimated the volume (V) of each cerebellar nucleus by multiplying the total area of each nucleus that is seen in the whole section of cerebellum (ΣA) with the mean section thickness (T), as shown in this formula V=∑A T [37]. The total number of cells in a nucleus was estimated by the formula N=NV V.
    Semiquantative Analysis of c-Fos-Immunoreactive Cells
    Analysis of the c-Fos-ir cells in the interposite nucleus of the cerebellum (IN) was performed using a previously described semiquantitative method [4]. In summary, photo- graphs of the IN were taken at ×4 magnification using an Olympus DP70 camera connected to an Olympus AX70 bright-field microscope (analySIS; Imaging System,
    Münster, Germany). The same light intensity and threshold settings of the camera were employed for all sections. For each animal, we selected two sections of the IN for quantification. The boundaries of the IN were delineated and the area measured. The counting of the c-Fos-ir cells was performed using the image analysis program ‘Image J’ (version 1.38, NIH, USA). A cell was counted as c-Fos-ir if its density was 65% higher than the mean background density of that section. Artefacts in the sections were excluded from analysis to ensure the accuracy of measurements. The number of c-Fos-ir cells per square millimetre was calculated.
    Statistical Analysis
    The data of the c-Fos counts were analysed using the Kruskal–Wallis test for non-parametric data. Non- parametric testing was chosen due to small group sizes. A value of p < 0.05 was considered significant. Post hoc analysis was performed using the Mann–Whitney test. Since we expected a deactivation in the stimulated groups, we used a one-tailed procedure.
    Histological Evaluation of the Electrode Localisation
    In all animals included in this study, the electrode tips were positioned correctly in the dlPAG and VMH, respectively (Fig. 1). No histological damage was observed except for the electrode trajectory with the standard haematoxylin– eosin staining, suggesting that stimulation with the current stimulation settings did not cause observable tissue damage.
    a dmPAG b dlPAG
    Fig. 1 Representative low-power photomicrographs of 30-μm-thick frontal sections from the brain of a rat subjected to stereotactic implantation of a concentric bipolar electrode to stimulate the dlPAG (a, scale bar = 250 μm) and VMH (b, scale bar = 500 μm). The tips of
    the electrodes are situated within the respective targets. Aquad aqueduct of Sylvius, dmPAG dorsomedial periaqueductal grey, dlPAG dorsolateral periaqueductal grey, 3V third ventricle, VMH ventrome- dial hypothalamus
    Cerebellum (2011) 10:61–69
    Behavioural Evaluation
    The intensity of the electrical current applied to the dlPAG and VMH of the animals was based on the threshold shown to induce escape behaviour. All rats showed ‘escape behaviour’ characterized by rigorous and aimless running. The current amplitudes necessary for inducing escape behaviour were significantly different between rats with dlPAG- and VMH-DBS (F's>351.13; p<0.00). The mean current density applied to the dlPAG to induce escape behaviour was approximately 90 μA and for the VMH, approximately 600 μA.
    Evaluation of the Number of c-Fos-Immunoreactive Cells
    There was a significant decrease in the amount of c-Fos-ir cells in the dentate nucleus (H2 = 6.343, p < 0.05; Figs. 2 and 3) and in the fastigial nucleus (H2 = 6.870, p < 0.05; Figs. 2 and 3) after DBS. As expected, we found a lower amount of c-Fos-ir cells in the dentate and fastigial nucleus of the stimulated rats compared to the control rats. Post hoc analysis showed that in the rats stimulated in the dlPAG, the difference with control rats was significant for both nuclei (DN, U = 1.00, r = −2.21; FN, U = 0.00, r = −2.45). When comparing VMH-stimulated rats to control rats, this difference was significant only in the nucleus dentatus (U = 1.00, r = −2.02); the difference in the fastigial nucleus showed a trend towards significance (U = 2.00, r = −1.73).
    Fig. 2 c-Fos expression in the dentate nucleus and in the fastigial nucleus of the cerebellum. Data are represented as means plus SEM showing control animals, animals stimulated in the dorsolateral periaqueductal grey (dlPAG) and animals stimulated in the ventrome- dial hypothalamus (VMH). The asterisk indicates a significant difference between groups. Note the significantly lower numbers of c-Fos immunoreactive neurons in the groups with animals showing panic attacks evoked by stimulation of the dlPAG and VMH
    There was no significant difference in the amount of c-Fos-ir cells between the stimulated groups (DN, U=7.00, r=−0.74; FN, U = 6.00, r = −0.98).
    Analysis of the interposite nucleus (IN) showed a similar trend towards decreased c-Fos expression in the stimulated rats compared to controls (PAG, U = 19.00, r = −1.04; VMH, U = 21.00, r = −1.37). Again, there was no difference between the stimulated groups (U = 36.00, r = −0.36).
    The objective of our experiment was to use electrical stimulation of the dlPAG and of the VMH in rats as a model of panic attack to study the involvement of the DCbN. We have found that panic-like behaviour was accompanied by a decrease of c-Fos-ir cells in the DCbN, indicating deactivation. c-Fos expression was significantly lower in the DN of both treatment groups and in the FN of the dlPAG DBS group when compared to controls. In the VMH DBS group, c-Fos expression in the FN was lower compared to controls, with a trend towards significance. Using a semiquantitative analysis, the IN showed a similar trend towards decreased c-Fos expression in the stimulated rats compared to controls. This should be considered a preliminary finding since the method of counting was not performed using stereological principles.
    There is anatomical and functional evidence supporting the role of the cerebellum in panic. Sakai et al. found a significantly higher glucose uptake in the cerebellum of patients with panic disorder compared to control patients [20]. After clinical improvement of these patients due to cognitive-behavioural therapy, the glucose uptake in cere- bellum had decreased [21]. Several groups found increased activation of the cerebellum after CCK-4-induced panic attacks in healthy subjects [24, 25]. This increase was not seen in these subjects during anticipatory anxiety. These studies were not initially designed to analyse changes in the cerebellum, and the changes found are usually unexpected. The findings are often simply mentioned as interesting or surprising, although several groups relate to the role of the cerebellum in fear conditioning as proposed by Sacchetti et al. [38]. Sacchetti et al. reviewed the evidence showing that the cerebellar vermis plays a role in the fear response and in fear conditioning, especially in fear consolidation. The most important changes are thought to take place at the level of the Purkinje cell (PC). It has been shown that several forms of fear conditioning lead to increased PC excitability and an increased firing rate of the PCs. In contrast, heterozygous Lurcher mice, which show early and complete apoptosis of cerebellar PCs, show reduced inhibition to anxiety- provoking aversive areas [39]. In summary, it seems that increased PC activity leads to more fear, and decreased or
    Cerebellum (2011) 10:61–69
    Control dlPAG-DBS VMH-DBS
    Fig. 3 Representative low-power photomicrographs of c-Fos expres- sion in a 10-μm-thick horizontal section of the deep cerebellar nuclei at Bregma level −6.10 mm (according to the stereotactic rat brain atlas of Paxinos and Watson of 1998) in a sham rat (a–c), a rat stimulated in the dlPAG (d–f), and a rat stimulated in the VMH (g–i). Panels a, d, and g were taken at ×2 magnification, and b, c, e, f, h, and i were taken at high-power magnification. The arrows point to a c-Fos-ir cell,
    absent PC activity to less fear. Functional imaging is designed to analyse activation changes in the cerebral cortex; therefore, it is likely that the activation seen in the cerebellum is also located in the cortex. Increased activa- tion found in functional imaging studies may therefore reflect the increased activity of the PCs. Since PCs are known to have an inhibitory action on the DCbN, these findings are in line with deactivation that we found in the DCbN in the stimulated rats which show panic-like behaviour.
    In the cerebellum, the vermis, projecting through the fastigial nucleus, seems to be the most important structure in fear and panic. In the cases described by Schmahmann et al., the vermis was always involved in patients with changes of affect [5]. In early experiments, vermal lesions were shown to attenuate a variety of fear behaviour,
    represented by a small dark dot. The inset in c shows a representative high-power photomicrograph of a c-Fos-ir cell. Scale bar for ×2 magnification power = 1 mm. dlPAG dorsolateral periaqueductal grey, VMH ventromedial hypothalamus, DBS deep brain stimulation, DN dentate nucleus of the cerebellum, FN fastigial nucleus of the cerebellum
    whereas vermal stimulation leads to increased fear-related responses [40, 41] Other animal research shows a selective role for the fastigial nucleus in heart-rate conditioning [40, 42, 43]. The vermis also contributes to consolidation of fear memory [44]. There is also evidence of a role of the interposite nucleus and the dentate nucleus in fear. In studies investigating several aspects of fear conditioning, animals with lesions of the dentate and interposite nucleus do not acquire an aversive conditioned response but acquire an appetitive conditioned response [45] and show unaltered heart-rate conditioning [42] and vocalisation indicative of unspecific fear [46]. Furthermore, there are clear bi- directional connections to the hypothalamus from (greatest to least concentration) the dentate nucleus, the interposite nucleus and the fastigial nucleus [47–50], supporting a role of all DCbN in autonomic processes, for example, those
    Cerebellum (2011) 10:61–69
    related to fear. In addition, the DCbN have been shown to project to several parts of the fear network: Teune et al. injected tracers in the DCbN in rats and documented projections to the PAG from the fastigial nucleus and the dentate nucleus and, in lesser degree, from the interposite nucleus [51]. Several groups have suggested projections from the fastigial nucleus to the amygdala, hippocampus, septal nuclei and the nucleus accumbens, based on behavioural changes following cerebellar stimulation or lesioning [38, 41, 52]. Berntson and Torello, for example, showed that hyperemotionality caused by septal lesions was largely attenuated by lesions of the fastigial nucleus [53]. An exact anatomical pathway has never been shown [54]. However, these behavioural changes indicate that the involvement of the cerebellum in fear does not merely consist of regulation of an autonomic visceromotor response but that there is a place for the cerebellum in the network regulating fear processing [38]. In a recent review, Stoodley and Schmahmann present a functional somatotopy of the non-motor functions of the cerebellum based on functional imaging [55]. They conclude that the vermis of the posterior lobe seems to be specifically related to emotional processing, whereas activation in the posterior cerebellar hemispheres may be related to the decision- making aspects of the tasks used in the experimental setting [55]. On the other hand, Timman et al. reviewed anatomical evidence for a role of the cerebellum in emotional and cognitive learning, and they conclude that with respect to fear, the vermis, projecting through the fastigial nucleus, contributes to the autonomic and somatic aspects, whereas the posterolateral cerebellar hemispheres, projecting through the dentate and interposite nucleus, play a role in the emotional content of fear processing [40].
    In summary, there is ample evidence that all cerebellar nuclei are involved in fear, in which the fastigial nucleus possibly mediates a different aspect of fear than the dentate nucleus and the interposite nucleus. This correlates with our findings in the present study showing a similar deactivation in the dentate nucleus and in the fastigial nucleus, and possibly also in the interposite nucleus. Increased fear is associated with increased PC activity in functional imaging studies, which is hypothesised to lead to inhibition of the DCbN and therefore of cerebellar output. We speculate that the cerebellum plays a role in regulating appropriate behaviour in response to any stimulus, and that a decreased cerebellar output may play a role in emergence of an inappropriate response, such as a panic attack. This decreased output may be in response to incorrect input, as suggested by Parvizi et al. [31]; however, we suggest that the cerebellum also plays a direct role in the selection of relevant information on which an adequate behavioural response is based, and that deactivation of the DCbN then leads to inappropriate behaviour by inhibiting this selection process.
    This study supports the hypothesis that the cerebellum is involved in panic attacks. Using DBS of the dlPAG and the VMH in rats as a model of a panic attack, we found that the activity of the DCbN as measured by c-Fos expression decreased significantly in the dentate nucleus and in the fastigial nucleus, and possibly also in the interposite nucleus. In a previous study, we showed that the DCbN are de- activated in rats showing increased impulsivity. In conclusion, deactivation of the cerebellum is associated with inappropriate behaviour such as panic and impulsivity. We suggest that the cerebellum plays a role in the selection of relevant informa- tion, thereby inhibiting such inappropriate behaviour.
    Acknowledgements The scientific work of Y. Temel has received funding from the Netherlands Organisation of Scientific Research (NWO-Veni grant) and the Dutch Brain Foundation (Hersenstichting Nederland). The authors are grateful to Mrs. Hellen Steinbusch for her technical assistance.
    Conflicts of interest The authors of this manuscript withhold no conflicts of interest.
    Open Access This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which per- mits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.
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    Neuroimage. Author manuscript; available in PMC 2011 May 24.

    Published in final edited form as:
    Neuroimage. 2011 March 1; 55(1): 389–400.
    Published online 2010 November 25. doi: 

    PMCID: PMC3100535
    NIHMSID: NIHMS292873

    Phasic and sustained fear in humans elicits distinct patterns of brain activity
    Ruben P. Alvarez,a,d,* Gang Chen,b Jerzy Bodurka,c,d Raphael Kaplan,a and Christian Grillona
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    Aversive events are typically more debilitating when they occur unpredictably than predictably. Studies in humans and animals indicate that predictable and unpredictable aversive events can induce phasic and sustained fear, respectively. Research in rodents suggests that anatomically related but distinct neural circuits may mediate phasic and sustained fear. We explored this issue in humans by examining threat predictability in three virtual reality contexts, one in which electric shocks were predictably signaled by a cue, a second in which shocks occurred unpredictably but never paired with a cue, and a third in which no shocks were delivered. Evidence of threat-induced phasic and sustained fear was presented using fear ratings and skin conductance. Utilizing recent advances in functional magnetic resonance imaging (fMRI), we were able to conduct whole-brain fMRI at relatively high spatial resolution and still have enough sensitivity to detect transient and sustained signal changes in the basal forebrain. We found that both predictable and unpredictable threat evoked transient activity in the dorsal amygdala, but that only unpredictable threat produced sustained activity in a forebrain region corresponding to the bed nucleus of the stria terminalis complex. Consistent with animal models hypothesizing a role for the cortex in generating sustained fear, sustained signal increases to unpredictable threat were also found in anterior insula and a frontoparietal cortical network associated with hypervigilance. In addition, unpredictable threat led to transient activity in the ventral amygdala–hippocampal area and pregenual anterior cingulate cortex, as well as transient activation and subsequent deactivation of subgenual anterior cingulate cortex, limbic structures that have been implicated in the regulation of emotional behavior and stress responses. In line with basic findings in rodents, these results provide evidence that phasic and sustained fear in humans may manifest similar signs of distress, but appear to be associated with different patterns of neural activity in the human basal forebrain.
    Keywords: Fear, Anxiety, Dorsal amygdala, BNST, Functional imaging, fMRI