REVIEW ARTICLE published: 17 October 2012 INTEGRATIVE NEUROSCIENCE doi: 10.3389/fnint.2012.00094 Modulation of physiological reflexes by pain: role of the locus coeruleus

Elemer Szabadi*

Psychopharmacology Section, Division of Psychiatry, University of Nottingham, Nottingham, UK

Edited by: The locus coeruleus (LC) is activated by noxious stimuli, and this activation leads to Mir Shahram Safari, Brain Science inhibition of perceived pain. As two physiological reflexes, the acoustic startle reflex Institute, RIKEN, Japan and the pupillary light reflex, are sensitive to noxious stimuli, this review considers Reviewed by: evidence that this sensitivity, at least to some extent, is mediated by the LC. The Marcelo F. Costa, Universidade de São Paulo, Brazil acoustic startle reflex, contraction of a large body of skeletal muscles in response to Wolfgang Liedtke, Duke University, a sudden loud acoustic stimulus, can be enhanced by both directly (“sensitization”) USA and indirectly (“fear conditioning”) applied noxious stimuli. Fear-conditioning involves the *Correspondence: association of a noxious (unconditioned) stimulus with a neutral (conditioned) stimulus Elemer Szabadi, (e.g., light), leading to the ability of the conditioned stimulus to evoke the “pain response”. Psychopharmacology Section, Division of Psychiatry, University of The enhancement of the startle response by conditioned fear (“fear-potentiated startle”) Nottingham, Room B109, Medical involves the activation of the amygdala. The LC may also be involved in both sensitization School, Queen’s Medical Centre, and fear potentiation: pain signals activate the LC both directly and indirectly via the Nottingham NG7 2UH, UK. amygdala, which results in enhanced motoneurone activity, leading to an enhanced e-mail: elemer.szabadi@ nottingham.ac.uk muscular response. Pupil diameter is under dual sympathetic/parasympathetic control, the sympathetic (noradrenergic) output dilating, and the parasympathetic (cholinergic) output constricting the pupil. The light reflex (constriction of the pupil in response to a light stimulus) operates via the parasympathetic output. The LC exerts a dual influence on pupillary control: it contributes to the sympathetic outflow and attenuates the parasympathetic output by inhibiting the Edinger-Westphal nucleus, the preganglionic cholinergic nucleus in the light reflex pathway. Noxious stimulation results in pupil dilation (“reflex dilation”), without any change in the light reflex response, consistent with sympathetic activation via the LC. Conditioned fear, on the other hand, results in the attenuation of the light reflex response (“fear-inhibited light reflex”), consistent with the inhibition of the parasympathetic light reflex via the LC. It is suggested that directly applied pain and fear-conditioning may affect different populations of autonomic neurones in the LC, directly applied pain activating sympathetic and fear-conditioning parasympathetic premotor neurones.

Keywords: pain, locus coeruleus, fear-conditioning, acoustic startle reflex, pupillary light reflex

INTRODUCTION the hypothalamic paraventricular nucleus (PVN) and tuberoin- The locus coeruleus (LC) has been implicated in a number fundibular area (Samuels and Szabadi, 2008a), and the LC has of physiological and psychological functions. The LC plays an been shown to be involved in stress responses associated with important role in the promotion and maintenance of arousal the activation of the hypothalamic-pituitary-adrenal axis (Plotsky (Robbins, 1984; Berridge, 2008; Carter et al., 2010; Berridge et al., et al., 1989; Valentino and Van Bockstaele, 2008; Ulrich-Lai and 2012). It is a wakefulness-promoting nucleus situated in a strate- Herman, 2009; Hermans et al., 2011). The LC plays an important gic position in the center of the arousal/sleep network, collecting role in the maintenance of muscle tone via an excitatory pro- information from both wakefulness- and sleep-promoting nuclei jection to motoneurones in the brainstem and the spinal cord in the network. Excitatory outputs from the LC project directly (Samuels and Szabadi, 2008a). Due to its extensive connections to the cerebral cortex and other wakefulness-promoting nuclei, to the amygdala, limbic system, and cerebral cortex, the LC has whereas inhibitory outputs project to sleep-promoting nuclei been implicated in a number of cognitive functions (in partic- (Samuels and Szabadi, 2008a,b). The LC is involved in autonomic ular, attention) and emotions (anxiety, mood) (Robbins, 1984; regulation: it contributes to sympathetic outflow by an excitatory Berridge and Waterhouse, 2003; Aston-Jones and Cohen, 2005; projection to preganglionic sympathetic neurones, and modulates Sara, 2009). The LC is also involved in the processing of pain parasympathetic activity via an inhibitory projection to parasym- signals and the modulation of pain sensation (see below). pathetic preganglionic neurones (Samuels and Szabadi, 2008a,b). It is known that two physiological reflexes, the acoustic LC activity also influences endocrine functions via connections to startle reflex, a somatic reflex, and the pupillary light reflex, an

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autonomic reflex, are both modulated by pain. In this review, of other neurotransmitters/neuromodulators has been suggested the possible involvement of the LC in the effect of pain on these on the basis of more indirect evidence, such as GABA (Pan et al., reflexes will be considered. Most of the experimental studies 2002), opiates (Pan et al., 2004), purines (Khakpay et al., 2010), quoted involve acute pain evoked by noxious stimulation; when and cannabinoids (Carvalho and Van Bockstaele, 2012). these studies are referred to, the stimulus is specified either in the text or in brackets after the reference. When the pain condition MODULATION OF PAIN BY THE LC was chronic (e.g., pathological or neuropathic pain), this is made The LC projects to pain-sensitive neurones of the trigeminal sen- clear in the text. sory nuclei, via the coeruleo-trigeminal pathway (Senba et al., 1981; Tsuruoka et al., 2003b) and the dorsal horn of the spinal PROCESSING OF PAIN BY THE LC cord, via the coeruleo-spinal pathway (Guyenet, 1980; Fritschy The nociceptive neurones of both the trigeminal sensory nuclei and Grzanna, 1990; Liu et al., 2007),andalsotothepain- in the brainstem and the dorsal horn of the spinal cord project, processing neurones of the somatosensory thalamus (Peschanski via the trigemino-thalamic and spino-thalmic pathways to the and Besson, 1984; Westlund et al., 1991; Voisin et al., 2005). Via somatosensory nucleus of the thalamus, a major pain-processing these modulatory pathways, the LC exerts an inhibitory influence subcortical relay nucleus. Pain signals from the thalamus reach on pain sensation [(Maeda et al., 2009) (hind paw inflamma- the somatosensory area of the cerebral cortex, the site of the tion)]; for reviews see Willis and Westlund (1997); Pertovaara highest level of pain processing, via the powerful thalamocor- and Almeida (2006); Ossipov et al. (2010). The LC can be acti- tical pathway (Kiernan, 2005). Apart from the somatosensory vated by noxious stimulation (see above), which leads to the thalamus, the LC is also an important subcortical relay nucleus inhibition of the pain evoked by a noxious stimulus, such as in the pain-processing system, channeling pain information to hind paw inflammation (Tsuruoka et al., 2003a; Maeda et al., the somatosensory cortex. Like the thalamus, the LC receives 2009). LC activation by direct electrical stimulation also evokes nociceptive inputs from both the trigeminal sensory nuclei and an anti-nociceptive effect [Margalit and Segal, 1979 (hot plate); the dorsal horn of the spinal cord (Craig, 1992). Furthermore, West et al., 1993 (noxious heat: foot withdrawal response)]. the LC projects to higher pain-processing structures, such as Interestingly, it has been reported that the anaesthetic gas nitrous the somatosensory thalamus, via the coeruleo-thalamic path- oxide activates the LC, and this effect has been implicated in the way (Peschanski and Besson, 1984; Westlund et al., 1991; Voisin analgesic effect of the drug [Sawamura et al., 2000 (tail flick, et al., 2005), and also directly to the cerebral cortex, via the hot plate)]. However, there are exceptions: the LC may facilitate, coeruleo-cortical pathway (Nieuwenhuys, 1985). Although the LC rather than attenuate, chronic neuropathic pain [Brightwell and innervates all areas of the neocortex, one of its main projection Taylor, 2009 (nerve injury-induced hyperalgesia)]. Furthermore, targets is the somatosensory cortex (Levitt et al., 1984; Gaspar the α2-adenoceptor agonist clonidine, a drug known to reduce et al., 1989). There is physiological evidence of the processing LC activity (Aghajanian and VanderMaelen, 1982; Williams et al., of pain signals, evoked by the electrical stimulation of the tooth 1985; Fernández-Pastor et al., 2005), has a paradoxical anal- pulp, by the LC, as revealed by recording the activity of single gesic effect [Sawynok and Reid, 1986 (tail flick); Sierralta et al., neurones in the somatosensory thalamus (Voisin et al., 2005). 1996 (acetic acid writhing test); Wang et al., 1998 (hot plate); There is extensive evidence showing that noxious stimula- Yoshikawa et al., 2001 (pain evoked by propofol injection in tion results in an increase in LC activity. Noxious stimuli evoke humans); Hauck et al., 2006 (electric shock to fingertips in an increase in the electrical activity of LC neurones, shown humans)]. However, as α2-adrenoceptors occur not only on LC by both extracellular [Kimura and Nakamura, 1985 (tail pinch, neurones, but at many other sites in the pain-processing/pain- air puff); Elam et al., 1986 (noxious heat); Rasmussen et al., modulating pathways (Pan et al., 2008; Ossipov et al., 2010), 1986 (pinch); Hirata and Aston-Jones, 1994 (foot shock)] and clonidine’s analgesic effect may not be mediated via the LC. intracellular [Sugiyama et al., 2012 (pinch)] recording. Painful Indeed, it has been reported that while clonidine decreases the stimulation leads to an increase in the expression of Fos, the activity of LC neurones in rats subjected to noxious stimula- protein product of the activation of the intermediate early gene tion by subcutaneously injected formalin, it activates dorsal horn c-Fos, a marker of neuronal activity [Bullitt, 1990 (noxious heat neurones in the spinal cord, implicating this latter action in the and cold, pinch); Pezzone et al., 1993 (electric shock); Palkovits analgesic effect of clonidine (Fukuda et al., 2006). et al., 1995 (subcutaneous formalin injection); Voisin et al., 2005 Conditioned pain modulation, also referred to as “diffuse (electric tooth pulp stimulation); Wang et al., 2009 (colon dis- noxious inhibitory controls,” involves the application of diffuse tension)]. Noxious stimuli evoke an increase in the release of relatively mild painful stimuli which lead to the attenuation of noradrenaline from the LC [Singewald et al., 1999 (air puff, the sensation of pain evoked by a localized strong noxious stim- noise stress); Kaehler et al., 2000 (tail pinch); Sajedianfard et al., ulus [Pertovaara and Almeida, 2006 (review); Lewis et al., 2012 2005 (subcutaneous formalin injection)]. A number of neuro- (noxious cold and pressure, ischaemic arm test in humans)]. transmitters and neuromodulators have been implicated in the This mechanism has been implicated in the analgesic effect of modulation of the activation of the LC by noxious stimuli. acupuncture [Bing et al., 1990 (noxious heat)]. It has recently There is direct evidence indicating the involvement of glutamate been reported that repeated injections of diluted bee venom, [Hayashida et al., 2010 (hind paw pressure)] and extracellular which lead to the attenuation of both acute pain evoked by a signal-regulated kinase (ERK) [Imbe et al., 2009 (subcutaneous noxious thermal stimulus and chronic (neuropathic) pain evoked formalin injection)]. Furthermore, the involvement of a number by the ligation of the sciatic nerve, activate the LC (Kang et al.,

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2012). Therefore the analgesic effect of conditioned pain modu- lation may involve the activation of the pain-inhibiting pathways arising from the LC. Indeed, duloxetine, a noradrenaline reuptake inhibitor which potentiates noradrenergic neurotransmission, enhances conditioned pain modulation [Yarnitsky et al., 2012 (noxious cold)], whereas dexmedetomidine, an α2-adrenoceptor agonist which reduces LC activity, inhibits it [Baba et al., 2012 (electric tooth pulp stimulation)]. Conditioned pain modulation is a form of sensory gating, and it may be analogous to “prepulse inhibition,” when a weak stimulus applied within a time window attenuates the effect of a strong stimulus (Perlstein et al., 2001). Indeed, it has been reported that pain sensation evoked by elec- tric shocks is subject to modulation by “prepulses” (Blumenthal et al., 2001). It is an intriguing possibility that the analgesic effect of prepulses, like that of conditioned pain modulation, may be related to the activation of the LC.

ACOUSTIC STARTLE REFLEX The acoustic startle reflex is the contraction of a large body of skeletal and facial muscles in response to a sudden loud auditory stimulus. The reflex pathway is simple, involving only four synapses (Figure 1). The reflex has been extensively stud- ied in both experimental animals and humans (Yeomans and Frankland, 1996; Koch and Schnitzler, 1997; Koch, 1999). In human subjects the response recorded is usually the contrac- tion of the orbicularis oculi muscle (eye-blink response) (Braff et al., 1978; Grillon et al., 1992; Kumari et al., 1996). The startle response is subject to sensory gating: a weak auditory stimulus FIGURE 1 | Central position of the locus coeruleus in relation to the applied within a time window prior to the index stimulus atten- acoustic startle reflex and pupillary light reflex pathways. Red: uates the response (“prepulse inhibition”) (Swerdlow et al., 1992; excitatory connections; blue: inhibitory connections. The acoustic startle Perlstein et al., 2001; Samuels et al., 2007). response is triggered by a sound stimulus activating auditory receptors in the cochlea. Auditory signals are transmitted via two nuclei of auditory As the final neurone in the reflex pathway is a motoneu- processing, the ventral cochlear nucleus and ventral nucleus of the lateral rone, which is under noradrenergic influence (Funk et al., 2000; lemniscus, to a relay nucleus in the pontine reticular formation, nucleus Heckman et al., 2009; Noga et al., 2011), the startle reflex is liable reticularis pontis caudalis, which projects directly to bulbar and spinal to be modulated by LC activity. Indeed, experimental lesioning of motoneurones. The startle response consists of the sudden synchronized the LC has been reported to result in a reduction in the amplitude contraction of a large array of facial and skeletal muscles. The locus coeruleus has a facilitatory influence on the motor neurones via an of the startle response (Adams and Geyer, 1981). excitatory noradrenergic output involving the stimulation of It is well documented that the LC sends excitatory projections, α1-adrenoceptors. Painful stimuli, via activation of the locus coeruleus can operating via the stimulation of α1-adrenoceptors, to motoneu- enhance the acoustic startle response (“sensitization”). The reflex rones in both the brainstem and the spinal cord (Samuels and response can also be enhanced by fear-conditioning via the amygdala. The Szabadi, 2008a). The noradrenergic projection to motoneurones lateral nucleus of the amygdala processes the association between aversive (painful) unconditioned (UCS) stimuli and neutral (e.g., light) conditioned plays an important role in the maintenance of muscle tone: when (CS) stimuli, and the arising conditioned fear signal is transmitted, via the LC activity is suspended, as during rapid eye movement sleep central nucleus of the amygdala, to the nucleus reticularis pontis caudalis, (Gottesmann, 2011) or attacks of cataplexy (Wu et al., 1999)total leading to the enhancement of the reflex response (“fear-potentiation”). atonia ensues (Peever, 2011). The amygdala also projects to the locus coeruleus, whose activation by conditioned fear contributes to the fear-potentiation of the acoustic startle LC neurones are under auto-regulation via inhibitory somato- α response. The pupillary light reflex is a parasympathetic autonomic reflex. dendritic 2-adrenoceptors that dampen neuronal firing as activ- Light signals stimulate photoreceptors in the retina which project, via ity increases (Huang et al., 2012). This mechanism may underlie melanopsin-containing intrinsically photosensitive retinal ganglion cells the observation that the LC “switches off” when very high firing (ipRGCs), to the pretectal nucleus, a parasympathetic premotor nucleus: frequencies are attained in response to stimulation (Carter et al., this leads to activation of the reflex pathway via the chain Edinger Westphal nucleus (preganglionic neurones) → ciliary ganglion (postganlionic 2010). Furthermore, it has been reported that narcolepsy is asso- neurones). The reflex response is the contraction of the smooth muscle ciated with an increase in the number of α2-adrenoceptors on LC fibres of the sphincter pupillae muscle, leading to pupil constriction neurones (Fruhstorfer et al., 1989):thiscouldleadtoincreased (miosis). The locus coeruleus has inhibitory influence on the preganglionic α auto-inhibition and the propensitiy of LC neurones to cease to fire neurones via a noradrenergic projection involving 2-adrenoceptors. As the when stimulated, as seen in attacks of cataplexy (Wu et al., 1999). locus coeruleus can be activated by the amygdala, it transmits conditioned fear signals to the Edinger Westphal nucleus, leading to the attenuation of Therefore, LC activity could be modified by experimental manip- the light reflex response by conditioned fear (“fear-inhibition”). ulation of central α2-adrenoceptors, which in turn could lead to

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changes in the acoustic startle response. Genetic manipulation of presence of chronic pain, such as functional abdominal pain in central α2C-adrenoceptors has been reported to be associated with children, can also lead to sensitization of the acoustic startle changes in the acoustic startle response in mice: targeted inac- response (Bakker et al., 2010). The degree of sensitization seems tivation of the gene encoding the receptor (α2C-KO) leading to to be related to the intensity of the noxious stimulus, such as elec- enhancement, and over-expression of the receptor to attenuation tric shock: stronger stimuli evoke more severe pain accompanied of the startle response (Sallinen et al., 1998). α2-Adrenoceptor by larger increases in the amplitude of the startle response (Duker agonists, such as clonidine, that are known to inhibit LC activity et al., 2004). (Aghajanian and VanderMaelen, 1982; Abercrombie and Jacobs, The phenomenon of sensitization is consistent with the 1987; Fernández-Pastor et al., 2005), attenuate the acoustic star- enhancement of LC activity evoked by the noxious stimulus, tle response in both animals (Davis et al., 1979) and humans which in turn would lead to increased motoneurone response at (Kumari et al., 1996; Abduljawad et al., 1997, 2001; Samuels et al., the final step in the acoustic startle response pathway (see above, 2007). On the other hand, the α2-adenoceptor antagonist yohim- and Figure 1). This mechanism may provide the physiological bine, a drug that increases LC activity (Ivanov and Aston-Jones, basis for the proposal of Davis (1989) that sensitization was a 1995; Crespi, 2009), has been reported to facilitate the acoustic simple “unlearned” or “unconditioned” response. However, this startleresponseinhumans(Morgan et al., 1993). While drugs view has been challenged (Richardson, 2000): since pain evoked targeted at α2-adrenoceptors are likely to have a direct effect on by a noxious stimulus is fear-inducing, sensitization may not LC activity, a number of drugs may modify LC activity indi- be fundamentally different from fear-potentiation (see, section rectly, by modulating excitatory and inhibitory inputs to the LC. “Effect of Conditioned Fear: Fear-Potentiation,” below). Indeed, Thus the wakefulness-promoting drug modafinil facilitates the there is evidence that the effect of pain on the acoustic startle acoustic startle response in humans (Samuels et al., 2007), prob- response may be susceptible to contextual factors, suggesting a ably by potentiating the dopaminergic excitation of LC neurones conditioning mechanism (Richardson and Elsayed, 1998). This (Hou et al., 2005). Indeed, the activation of the LC by modafinil may explain, that in some situations sensitization could not be has been demonstrated by fMRI in human subjects (Minzenberg observed [Horn et al., 2012a,b (noxious heat in humans)], or that et al., 2008). On the other hand, diazepam has been shown to the noxious stimulus inhibited, rather than potentiated, the star- reduce the amplitude of the acoustic startle response in both ani- tle response [Sorenson and Swerdlow, 1982 (tail pinch in rats); mals (Berg and Davis, 1984) and humans (Abduljawad et al., Tavernor et al., 2000 (noxious cold in humans)]. 1997, 2001). This effect of diazepam has been attributed to a The overlap between the mechanisms underlying sensitization reduction in LC activity associated with sedation (Samuels et al., and fear-potentiation is further strengthened by the observation 2007; Samuels and Szabadi, 2008b). However, it should be noted that the amygdala, a structure essential for fear-conditioning (see that the effect of diazepam on LC activity is likely to be indirect, below, and Figure 1), is also involved in sensitization (Hitchcock since GABA receptors in the LC have been reported to be insen- et al., 1989; Fendt et al., 1994b; Krase et al., 1994). The involve- sitive to diazepam (see section “Effect of Pain on Pupil Diameter: ment of the LC in sensitization is rather complex. Pain may Reflex Dilation,” below). As the level of arousal is closely associ- activate the LC both directly (see above) and indirectly via the ated with LC activity (Samuels and Szabadi, 2008b), drugs known amygdala, which in turn projects to the LC (Figure 1)(Samuels to facilitate the acoustic startle response, in general, are stimu- and Szabadi, 2008a; Reyes et al., 2011); LC activation would lants, whereas drugs inhibiting it are sedatives (Samuels et al., lead to the potentiation of motoneurone activity, resulting in the 2007). enhancement of the startle response. There is also a reciprocal The acoustic startle response has an autonomic component: connection between the LC and the amygdala: the LC does not the auditory stimulus also evokes a sympathetic response, includ- only receive an input from the amygdala, but it also projects ing increases in blood pressure and heart rate (Baudrie et al., 1997; to it (Samuels and Szabadi, 2008a). The reciprocal connection Holand et al., 1999; Eder et al., 2009) and sweat gland activity between the LC and the amygdala provides the basis for a posi- (Samuels et al., 2007). Interestingly, the autonomic component of tive feed-back mechanism: by projecting to the amygdala, the LC the startle reflex, like the motor component (see above), is sub- can enhance its own activation by this structure. Indeed, it has ject to prepulse modulation (Samuels et al., 2007; Eder et al., been shown that noradrenaline release in the amygdala is involved 2009). Although the exact connections of this “sensorysympa- in the sensitization of the acoustic startle response (Fendt et al., thetic reflex” are not known, it is likely that a number of premotor 1994b). Finally, LC activation by pain may increase the sensitivity sympathetic nuclei, including the ventrolateral medulla (Holand of the startle reflex to auditory stimuli via an excitatory LC pro- et al., 1999), the LC and the hypothalamic paraventricluar nucleus jection to cochlear nuclei (Kromer and Moore, 1976; Ebert, 1996; (Samuels et al., 2007)areinvolved. Gómez-Nieto et al., 2008), resulting in enhancement of the reflex response. EFFECT OF PAIN: SENSITIZATION It has been reported that acutely applied noxious stimuli,such EFFECT OF CONDITIONED FEAR: FEAR-POTENTIATION as foot shocks in rats (Davis, 1989; Fendt et al., 1994a,b; Fear-conditioning is based on associative learning (“Pavlovian Krase et al., 1994) and noxious heat in humans (Grombez conditioning”): pairing of a noxious (“unconditioned”) stimu- et al., 1997), increase the amplitude of the acoustic startle lus (US) with a neutral (“conditioned”) stimulus (CS), results in response (“sensitization”) (for reviews, see Koch and Schnitzler, the development of the ability of the CS to evoke the response 1997; Fendt and Fanselow, 1999; Koch, 1999). Interestingly, the totheUS(seee.g.,Fendt and Fanselow, 1999). There is a large

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body of evidence that the acoustic startle response is subject to PUPILLARY LIGHT REFLEX modulation not only by pain (see section “Modulation of Pain The pupil is an aperture in a diaphragm, the iris, whose function by the LC”, above), but also by conditioned fear arising from is to control the amount of light reaching the retina. The iris con- the prior pairing of a painful stimulus with a neutral stimulus tains two smooth muscles, the sphincter (or constrictor) pupil- (e.g., light). Conditioned fear leads to the enhancement of the lae, and the dilator pupillae. The two muscles receive opposing acoustic startle response (“fear-potentiation”) (Davis, 1992; Davis parasympathetic and sympathetic innervations, and thus pupil et al., 1993; Koch and Schnitzler, 1997; Fendt and Fanselow, 1999; diameter at any time reflects the intricate balance between the two Koch, 1999). Fear-potentiation of the acoustic startle reflex has output systems (Samuels and Szabadi, 2008a,b). The pupillary been studied extensively in both animals (Davis et al., 1993; Koch, light reflex, an autonomic reflex, consists of the constriction of the 1999) and humans (Grillon et al., 1991; Bitsios et al., 1999a,b; pupil in response to a light stimulus. The reflex pathway is shown Scaife et al., 2005; Hubbard et al., 2011). The unconditioned stim- in Figure 2. Three photoreceptors are involved: rods and cones ulus used in these experiments is usually electric shock: foot shock that project to a subgroup of retinal ganglion cells (“intrinsically in animals and shock to the volar surface of the wrist in humans. It photosensitive ganglion cells, ipRGCs”) containing the photopig- has been shown that the brain structure processing the association ment melanopsin. The ipRGCs constitute the third photoreceptor between US and CS is the lateral nucleus of the amygdala, which (Kawasaki and Kardon, 2007). The ipRGCs mediate not only is connected to the central nucleus of the amygdala, the structure the pupillary light reflex, but also all other “non-image-forming” projecting to the caudal pontine reticular nucleus, a major relay (NIF) visual functions, such as the regulation of circadian nucleus in the acoustic startle reflex pathway (Figure 1)(Davis, rhythms, sleep/arousal and autonomic/endocrine activity. The 1992; Davis et al., 1993). The activity of neurones in the amyg- ipRGCs give rise to two pathways: one to the olivary pretectal dala is susceptible to modulation by conditioned fear, as shown by increases in their firing rate [Pascoe and Kapp, 1985 (electric shock)] and in the expression of Fos in this structure (Davis et al., 1993). As discussed above (see “Modulation of Pain by the LC”), the central nucleus of the amygdala also projects to the LC, which in turn influences the activity of the motor neurone pool, the final step in the reflex pathway. There is evidence that the activity of the LC, like that of the amygdala, is subject to modulation by fear con- ditioning: neutral stimuli, previously associated with painful ones (i.e., electric shocks), increase LC activity, as shown by increases both in neuronal firing rate (Rasmussen and Jacobs, 1986)and Fos expression (Pezzone et al., 1993; Ishida et al., 2002; Liu et al., 2003). A number of drugs can modify the fear-potentiated startle response (Davis, 1992; Davis et al., 1993; Fendt and Fanselow, 1999). It is likely that different drugs may act at specific sites within the reflex pathway. It is well documented that benzodi- azepines attenuate or even block the response, both in animals (Davis, 1979; Berg and Davis, 1984; Hijzen and Slangen, 1989; Davis et al., 1993) and humans (Patrick et al., 1996; Bitsios et al., 1999a; Graham et al., 2005; Scaife et al., 2005). Although FIGURE 2 | Neuronalnetworkmediating theeffect oflight on pupil these drugs may act at a number of sites in the brain, it is diameter. Red: excitatory connections, blue: inhibitory connections. Hypothalamic nuclei: SCN, suprachiasmatic nucleus; PVN, paraventricular likely that an action at the level of the amygdala is relevant. nucleus; DMH, dorsomedial hypothalamus; autonomic premotor nuclei: It has been reported that diazepam can selectively block the OPN, olivary pretectal nucleus; LC, locus coeruleus; parasympathetic acquisition but not the expression of fear potentiation (Scaife nucleus/ganglion: EW, Edinger Westphal nucleus; GC, ganglion ciliare; et al., 2005, 2007), suggesting interference with the process- sympathetic nucleus/ganglion: IML, intermedio-lateral column of spinal cord; ing of the association between US and CS, which is known to SCG, superior cervical ganglion. Neurotransmitters: Glu, glutamate; GABA, γ-amino-butyricacid;VP,vasopressin; Ox, orexin; ACh, acetylcholine; NA, be localised in the amygdala (Davis, 1992; Davis et al., 1993). noradrenaline. Adrenoceptors: α1 , excitatory and α2 , inhibitory. Pupil diameter The α2-adrenoceptor agonist clonidine is also a potent inhibitor reflects the relationship between two opposing smooth muscles, the dilator of the fear-potentiated startle response (Davis et al., 1979). As pupillae, innervated by the sympathetic, and sphincter (constrictor) pupillae a major action of clonidine is the inhibition of LC activity (innervated by the parasympathetic). The locus coeruleus functions as both a (Aghajanian and VanderMaelen, 1982; Abercrombie and Jacobs, sympathetic and a parasympathetic premotor nucleus: it stimulates preganglionic sympathetic neurones in the IML and inhibits preganglionic 1987; Fernández-Pastor et al., 2005), the inhibition of the fear- parasympathetic neurones in the EW. The pupillary light reflex is a potentiated startle by this drug corroborates the evidence about parasympatheticreflex:lightsignals fromtheretinastimulate thechain OPN→ the involvement of the LC in fear-potentiation. Interestingly, the EW→ GC, leading to pupil constriction. Light also has an indirect effect on sympatheticactivityviatheSCN:sympatheticactivityisinhibitedviaaninhibitory α2-adrenoceptor antagonist yohimbine, a drug known to stimu- late the LC (Ivanov and Aston-Jones, 1995, Crespi, 2009), has the output to the PVN. Light-evoked sympatho-inhibition, however, is likely to be attenuatedbysympatho-excitation mediatiedviatheSCN→DMH→LCroute. opposite effect: it facilitates fear-potentiation (Davis et al., 1979).

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nucleus (OPN), a preomotor nucleus in the parasympathetic light causes pupil dilation (mydriasis). This latter effect is likely to reflex pathway, and another one (retino-hypothalamic tract) to reflect the activation of postsynaptic inhibitory α2-adrenoceptors two hypothalamic nuclei, the suprachiasmatic nucleus (SCN) and on EWN neurones in preference to the stimulation of presy- the ventrolateral preoptic nucleus (VLPO) (Figures 2 and 3)(Lu naptic autoreceptors on LC neurones (Samuels and Szabadi, et al., 1999; Gooley et al., 2003; Güler et al., 2008). The SCN is 2008b). responsible for the generation of circadian rhythms and modula- tion of autonomic and endocrine functions (Kalsbeek et al., 2000, MODULATION OF LC ACTIVITY BY LIGHT 2006; van Esseveldt et al., 2000), whereas the VLPO is a major Interestingly, light does not only evoke the light reflex, but also sleep-promoting nucleus (Lu et al., 1999; Samuels and Szabadi, influences the sympathetic output to the iris. Light, by inhibit- 2008b). ing the PVN, a major sympathetic premotor nucleus, via the The pupillary light reflex is a parasympathetic reflex: the OPN SCN, exerts an inhibitory effect on the activity of preganglionic projects to the preganglionic parasympathetic cholinergic neu- sympathetic neurones in the IML of the upper thoracic (T1- rones located in the Edinger-Westphal nucleus (EWN) of the T3) spinal cord, synapsing in the superior cervical ganglion midbrain; the EWN innervates the postganglionic cholinergic (Kalsbeek et al., 2000; Perreau-Lenz et al., 2003): this effect leads neurones in the ciliary ganglion; the postganglionic neurones to pupil constriction (Passatore, 1976; Passatore and Pettorossi, innervate the constrictor pupillae muscle. 1976; Szabadi et al., 2010) and inhibition of melatonin synthe- The sympathetic innervation of the iris does not directly par- sis (Nishino et al., 1976; Kalsbeek et al., 1999; Zeitzer et al., ticipate in the light reflex: its main function is the adjustment of 2000). However, light may also have a stimulatory effect on pupil diameter to the level of background illumination. The sym- sympathetic activity, by stimulating the LC via the SCN and pathetic preganglionic neurones are located in the intermedio- the dorso-medial hypothalamus (DMH) (Figure 3)(Aston-Jones lateral column of the cervico-thoracic spinal cord, and project to the noradrenergic postganglionic neurones in the superior cer- vical ganglion, which innervate the dilator pupillae muscle. A number of premotor nuclei modulate the activity of the pregan- glionic sympathetic neurones, of which the PVN of the hypotha- lamus and the LC are the most important (Figure 2)(Samuels and Szabadi, 2008a).

MODULATION OF THE PUPILLARY LIGHT REFLEX BY THE LC The LC plays a dual role in pupillary control: it contributes to sympathetic outflow via an excitatory projection to preganglionic sympathetic neurones, and it also modulates the parasympa- thetic output to the iris via an inhibitory connection to the EWN. The close relationship between LC activity and pupil- lary function is illustrated by the observation that fluctuations FIGURE 3 | Neuronal network mediating the dual effect of light on in the firing rate of LC neurones are closely paralleled by fluc- arousal. Nuclei: yellow: wakefulness-promoting, purple: sleep-promoting. tuations in the diameter of the pupil (Aston-Jones and Cohen, Connections: red: excitatory, blue: inhibitory. Hypothalamic nuclei: SCN, suprachiasmatic nucleus; DMH, dorso-medial hypothalamus; LH, lateral 2005). hypothalamic area; VLPO, ventrolateral preoptic nucleus; TMN, Via its projection to the EWN, the LC inhibits the pupil- tuberomamillary nucleus; brainstem nucleus: LC, locus coeruleus. lary light reflex. It has been shown that anxiety, an emotional Neurotransmiters: Glu, glutamate; Ox, orexin; NA, noradenaline; H, state known to be associated with LC activation (Millan, 2003), histamine. Light reaching the retina has a sleep-promoting effect via the excitatory output of melanopsin-containing intrinsically photosensitive leads to attenuation of the pupillary light reflex response (Bakes retinal ganglion cells (ipRGCs) to the VLPO, the major sleep-promoting et al., 1990). Similarly, drugs that are likely to increase nora- nucleus. GABAergic inhibitory neurones in the VLPO project to the cerebral drenergic output to the EWN (e.g., noradrenaline re-uptake cortex, and two major wakefulness-promoting nuclei, the TMN and LC. inhibitors), decrease the amplitude of the light reflex response Light also evokes a wakefulness-promoting effect via the SCN which can (Theofilopoulos et al., 1995; Bitsios et al., 1999b; Szabadi and stimulate orexinergic and glutamatergic neurones in the DMH that project α to two wakefulness-promoting nuclei, the LC and LH. There is a reciprocal Bradshaw, 2000). On the other hand, the 2-adrenoceptor ago- inhibitory connection between the LC and the VLPO: GABAergic neurones nist clonidine, a drug “switching off” LC activity by stimulat- in the VLPO inhibit the LC, and noradrenergic neurones in the LC inhibit the ing inhibitory autoreceptors on LC neurones (Aghajanian and VLPO via the stimulation of α2-adrenoceptors. The LC also stimulates the α VanderMaelen, 1982; Abercrombie and Jacobs, 1987; Fernández- cortex and the TMN via excitatory outputs involving 1-adrenoceptors. The Pastor et al., 2005), causes pupil constriction (miosis), due orexinergic neurones of the LH send excitatory outputs to the LC and the cerebral cortex. The overall effect of light on arousal depends on the partly to the inhibition of sympathetic outflow and partly relationship between the two light-sensitive arousal systems. In nocturnal to the removal of the noradrenergic inhibition of the EWN animals light is sleep-promoting due to the predominant effect of the (Samuels and Szabadi, 2008b). However, it should be noted activation of the VLPO by light. In diurnal animals, on the other hand, the that the effect of clonidine depends on the species studied: sleep-promoting effect of VLPO activation by light, is likely to be while in diurnal animals (man, dog, and rabbit) clonidine superseded by the wakefulness-promoting influence of the activation of the LCandLHviatheSCN→ DMH → LC/LH route. causes miosis, in nocturnal animals (cat, rat, and mouse) it

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et al., 2001; Aston-Jones, 2005; Gonzalez and Aston-Jones, 2006). patients (Walch et al., 2005), consistent with enhanced LC Indeed, it has been shown by fMRI in humans that short wave- activity. length (blue) light, that preferentially stimulates the melanopsin- containing photoreceptors of ipRGCs (Kawasaki and Kardon, EFFECT OF PAIN ON PUPIL DIAMETER: REFLEX DILATION 2007), causes activation in a brain stem area corresponding to “Any sensory stimulus (with the exception of light) can elicit the LC (Vandewalle et al., 2007). Interestingly, the overall effect pupillary dilation” (Loewenfeld, 1993), and noxious stimuli are of light on sympathetic activity is inhibitory at the upper tho- no exception. It is well documented that painful stimuli dilate racic (T1–T3) levels of the spinal cord, whereas it is stimulatory the pupil in both animals and humans. In animals, the nox- at the levels of lower thoracic segments, leading to stimulation ious stimulus used was electric stimulation of the sciatic nerve of cardiovascular activity (Michimori et al., 1997; Scheer et al., (Koss et al., 1984; Hey et al., 1985; Koss, 1986; Hey and Koss, 1999; Cajochen et al., 2005), and corticosterone secretion from 1988; Yu and Koss, 2003, 2004), and in humans either noxious the adrenals (Niijima et al., 1992; Ishida et al., 2005; Hatanaka cold (“cold pressor test”) (Tassorelli et al., 1995; Tavernor et al., et al., 2008). The effect of light on cardiovascular activity seems to 2000; Hou et al., 2007) or electric shock (Chapman et al., 1999; be species-dependent: while it is stimulatory in diurnal animals, Larson and Talke, 2001; Yang et al., 2003; Walter et al., 2005; it is inhibitory in nocturnal animals (Scheer et al., 2001), con- Oka et al., 2007). As noxious stimulation activates the LC (see sistent with the close association of cardiovascular activity with section “Processing of Pain by the LC,” above), and the LC is inti- the level of arousal (see below). The spinal-segment-related effect mately involved in pupillary control (see section “Modulation of of light on sympathetic outflow, which conforms to the principle the Pupillary Light Reflex by the LC” and Figure 2), it is likely that of sympathetic organization according to “tissue-specific sympa- reflex dilation of the pupil (mydriasis) evoked by painful stimuli is thetic output pathways” (Morrison, 2001), has been attributed to mediated by the LC. Indeed, it has been reported that reflex pupil the operation of region-specific specialization in the SCN (Scheer dilation to noxious stimuli is abolished following central nora- et al., 2003). drenaline depletion by reserpine or alpha-methyl-para-tyrosine The modulation of LC activity by light is likely to be dif- (Koss et al., 1984). An increase in LC activity can lead to pupil ferent between nocturnal and diurnal animals, as highlighted dilation in two ways: by increasing sympathetic outflow to the iris, by the effect of light on arousal: in nocturnal animals, light via an excitatory output to sympathetic preganglionic neurones is sleep-promoting (Altimus et al., 2008; Tsai et al., 2009), in the spinal cord, and by inhibiting parasympathetic output, whereas in diurnal animals, including man, is wakefulness- via an inhibitory connection to parasympathetic preganglionic promoting (Cajochen et al., 2005; Lockley et al., 2006; Revell neurones in the EWN. et al., 2006). In both nocturnal and diurnal animals light There is direct evidence that painful stimulation increases has a dual effect: promoting sleep via the activation of the impulse flow in sympathetic fibres innervating the iris [Passatore, VLPO, and wakefulness via the activation of the SCN-MDH-LC 1976 (pinch, corneal touch)]. The importance of sympathetic circuit (Figure 3), the overall effect depending on the relation- activation in reflex pupil dilation to painful stimuli has been ship between the two opposite influences. Therefore in diur- demonstrated in humans. Firstly, the cold pressor test (plung- nal animals the wakefulness-promoting effect of the activation ing one hand into ice cold water), which constitutes an intense of the SCN-MDH-LC circuit is likely to supersede the sleep- painful stimulus (Yarnitsky and Ochoa, 1990)andisapow- promoting effect arising from the activation of the VLPO. It erful sympathetic activator (Seals, 1990), evokes pupil dilation is an intriguing possibility that the greater sensitivity of presy- (Tassorelli et al., 1995; Tavernor et al., 2000; Hou et al., 2007). naptic α2-adrenoceptors on LC neurones to clonidine, com- Secondly, reflex dilation of the pupil can be antagonized by pared to postsynaptic receptors, in diurnal animals (see section topical application of α1-adrenoceptor antagonists (e.g., thymox- “Modulation of the Pupillary Light Reflex by the LC,” above), may amine: Tassorelli et al., 1995;dapiprazole:Yang et al., 2003; Hou be related to the more pronounced activation of the LC by light et al., 2007) to the cornea: these drugs would block the effect in these species. of noradrenaline released by sympathetic stimulation. Thirdly, The activation of the LC by light may have implications the α2-adrenoceptor agonist dexmedetomidine has been reported for the processing and modulation of pain signals. It has been to reduce reflex dilation of the pupil (Larson and Talke, 2001), reported that diurnal animals, including man, are less sensi- consistent with a central sympatholytic effect resulting from the tive to pain during day time (Rigas et al., 1990; Göbel and inhibition of the LC by dexmedetomidine (Chiu et al., 1995). Cordes, 2005) whereas the opposite seems to be the case in As benzodiazepines have been reported to inhibit sympathetic nocturnal animals (Kavaliers and Hirst, 1983; Kavaliers et al., activity (Marty et al., 1986; Ikeda et al., 1994; Kitajima et al., 1984; Yoshida et al., 2003). The time-of-day-related variations 2004), it would be expected that these drugs, like α2-adrenoceptor in pain sensitivity have been attributed to circadian variations agonists, would antagonize reflex dilation of the pupil. However, in endorphin synthesis (Kavaliers and Hirst, 1983; Rasmussen in a study examining the effect of diazepam on pupillary and and Farr, 2003). However, they may also reflect diurnal vari- cardiovascular functions, diazepam failed to attenuate reflex dila- ations in LC activity: in diurnal animals the LC is maximally tion of the pupil, while it antagonized the increase in systolic active during day time, and LC activity is known to be associ- blood pressure, evoked by the cold pressor test (Hou et al., ated with an analgesic effect (see section “Processing of Pain by 2007). Interestingly, a similar dissociation between the effects of the LC,” above). Furthermore, it has been reported that expo- diazepam on pupil dilation and increase in blood pressure evoked sure to bright sunlight reduces post-operative pain in surgical by central sympathetic stimulation has been reported in cats:

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pupil dilation remained unaffected while the blood pressure rise the Pupillary Light Reflex by the LC” and “Modulation of LC was antagonized (Sigg and Sigg, 1969). These observations sug- Activity by Light,” above). gest that different sections of the sympathetic nervous system, one diazepam-insensitive and another one diazepam-sensitive, EFFECT OF CONDITIONED FEAR ON LIGHT REFLEX: FEAR INHIBITION may be responsible for mediating pupillary and cardiovascular As conditioned fear potentiates the acoustic startle reflex, and the responses (Sigg et al., 1971). The diazepam-insensitive system, activation of the LC is likely to contribute to this effect (see section responsible for pupillary control, is likely to involve the LC, since “Effect of Conditioned Fear: Fear-Potentiation,” above), it could LC neurones, although they contain GABAA receptors (Kaur be predicted that conditioned fear may also affect the pupillary et al., 1997; Chen et al., 1999), have been reported to be insen- light reflex which is under inhibitory noradrenergic control from sitive to diazepam (Chen et al., 1999). On the other hand, a likely the LC (see section “Modulation of the Pupillary Light Reflex by candidate for the diazepam-sensitive system is the one originating the LC,” above). This prediction seemed to be supported by the in the PVN. The role of the PVN in cardiovascular control is well observation that patients suffering from general anxiety disorder established (Coote, 2005; Li and Pan, 2007; Womack et al., 2007; had attenuated light reflex responses, with relatively little change Nunn et al., 2011), and it has been shown that sympathetic pre- in resting pupil diameter, compared to healthy controls (Bakes motor neurones in the PVN are inhibited by diazepam (Zahner et al., 1990). The reduction in the amplitude of the light reflex et al., 2007). response in the anxious patients could be interpreted as the con- While noxious stimulation in humans results in the activation sequence of enhanced LC activity associated with anxiety (Millan, of the sympathetic outflow to the iris, there is no evidence of any 2003). alteration in the parasympathetic output to the iris. Indeed, it has Using a protocol identical to that which had been applied in a been reported that the pupillary light reflex response, a sensitive study of the effect of conditioned fear on the acoustic startle reflex index of LC activity (see section “Effect of Pain: Sensitization,” in human volunteers, using electric shock as the conditioned above), remains unaffected by noxious stimulation (Tavernor stimulus (Grillon et al., 1991), it was shown that conditioned et al., 2000; Hou et al., 2007). Therefore, pupillary reflex dilation fear caused an increase in pupil diameter and a reduction in the in humans seems to be a pure sympathetic response. Interestingly, amplitude of the light reflex response, associated with increases the same pattern seems to apply to rabbits, in whom no parasym- in subjective ratings of alertness and anxiety (Bitsios et al., 1996). pathetic contribution to pupillary reflex dilation could be demon- In a subsequent experiment, the effect of conditioned fear on the strated, and thus the response appears to be mediated entirely by two reflexes was compared within the same subjects and sessions. the sympathetic (Yu and Koss, 2003, 2004). Conditioned fear had opposite effects on the two reflexes: the On the other hand, a different pattern has been reported in acoustic startle reflex was potentiated, while the pupillary light cats and rats (Koss et al., 1984; Hey et al., 1985; Koss, 1986; Hey reflex was inhibited (Bitsios et al., 1999a). Figure 1 illustrates the and Koss, 1988). In these species, pupil dilation evoked by nox- position of the LC in modulating both the acoustic startle reflex ious stimulation is preserved after cutting the sympathetic nerve and the pupillary light reflex. In the case of both reflexes, the innervating the dilator muscle of the iris; the α2-adrenoceptor amygdala, the structure processing the association between US agonist clonidine, like noxious stimulation, dilates the pupil; the (e.g., pain) and CS (e.g., light) plays a central role: activation α2-antagonist yohimbine antagonizes the pupil dilatory effects of of the amygdala leads to the activation of the LC. LC activa- both clonidine and noxious stimulation. On the basis of these tion, however, has opposite effects on the two reflexes: while it observations, Koss and his colleagues concluded that reflex dila- has a facilitatory effect on the acoustic startle reflex by enhancing tion of the pupil was mediated by noradrenergic inhibition of the the noradrenergic excitation of motoneurones via the stimulation preganglionic parasympathetic neurones in the EWN, with little of α1-adrenoceptors, it has an inhibitory effect on the pupillary contribution by the sympathetic (Koss, 1986). light reflex by enhancing the noradrenergic inhibition of pregan- The apparent species difference in the mediation of reflex glionic parasympathetic neurones in the EWN via the stimulation dilation of the pupil evoked by noxious stimuli is consistent of α2-adrenoceptors. with the suggestion that separate populations of LC neurones Like in the case of the fear-potentiation of the acoustic star- may function as sympathetic and parasympathetic premotor neu- tle reflex (Grillon et al., 1991; Davis et al., 1993), anxiety plays rones (Samuels and Szabadi, 2008a). Thus in diurnal animals an important role in the inhibition of the pupillary light reflex (rabbit, man) painful stimuli my activate predominantly the sym- by conditioned fear. The anticipation of an aversive stimulus (i.e., pathetic premotor neurones in the LC, whereas in nocturnal pain-induced by an electric shock), leading to the inhibition of animals (rat, cat) they may activate predominantly the parasym- the light reflex, has been reported to be associated with increases pathetic premotor neurones. This species difference in the effect in subjective ratings of anxiety (Bitsios et al., 1996, 1999a,b, 2004). of painful stimuli on the LC is paralleled by species differ- Furthermore, the size of the effect of conditioned fear on the ences in the effect of light on arousal (wakefulness-promoting in pupillary light reflex has been reported to be related to the pre- diurnal animals, sleep-promoting in nocturnal animals) and car- existing level of anxiety, subjects with higher levels of “state” diovascular activity (stimulatory in diurnal animals, inhibitory anxiety showing larger effects (Bitsios et al., 2002). in nocturnal animals), and is also reflected in the site of action Drugs that are known to have effects on the fear-potentiated of α2-adrenoceptor agonists on central noradrenergic neurones acoustic startle reflex (see section “Effect of Conditioned Fear: (predominantly presynaptic in diurnal animals, predominantly Fear-Potentiation”, above), also modify the fear-inhibited light postsynaptic in nocturnal animals) (see sections “Modulation of reflex. It has been reported that both clonidine (Bitsios et al.,

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1998a) and diazepam (Bitsios et al., 1998b, 1999a) can antago- LC activation by the amygdala enhances the noradrenergic nize the effect of conditioned fear on the pupillary light reflex. As facilitation of motoneurone activity, leading to augmentation of discussed in relation to the fear-potentiated startle reflex, cloni- the potentiation of the acoustic startle reflex, arising from the dine may act directly on the LC, whereas the most likely site of stimulation of the pontine relay nucleus in the reflex pathway. action of diazepam is the amygdala. LC activation by the amygdala leads to the attenuation of the Conditioned fear has a dual effect on the pupil: it causes a pupillary light reflex due to enhancement of the noradrener- small increase in pupil diameter together with the inhibition of gic inhibition of the preganglionic parasympathetic neurones in thelightreflexresponse(Bitsios et al., 1996, 1999a,b, 2004). There the light reflex pathway. Interestingly, there seems to be some are indications that the two effects may be mediated by separate selectivity in the action of pain applied directly or indirectly, via mechanisms: the increase in pupil diameter can be evoked by the fear conditioning, on premotor autonomic neurones in the LC: anticipation of a neutral (e.g., acoustic) stimulus, and is accompa- while directly applied pain stimulates the sympathetic premotor nied by an increase in the level of alertness, whereas the inhibition neurones, leading to pupil dilation, in diurnal animals, fear- of the light reflex can be evoked by the anticipation of an aversive conditioning stimulates the parasympathetic premotor neurones, stimulus, and is accompanied by an increase in anxiety (Bitsios leading to attenuation of the light reflex response. et al., 2004). These observations suggest that the effect of condi- There are some clinical conditions, usually involving pain, tioned fear on pupil diameter may involve mainly the activation fear/anxiety and/or stress, that may be associated with abnor- of sympathetic premotor neurones in the LC, that are also closely malities of the two reflexes. Furthermore, it is likely that these associated with the modulation of arousal. On the other hand, the abnormalities reflect altered LC activity. Post-traumatic stress dis- effect of conditioned fear on the light reflex may mainly be due to order (PTSD) is “a chronic, debilitating psychiatric disorder that the activation of parasympathetic premotor neurones in the LC, can follow exposure to extreme stressful experiences” (Stam, which may be closely associated with anxiety. This hypothesis is 2007). Cardinal features of the syndrome include hyperarousal, supported by the observation that the α1-adrenoceptor antago- increased startle responses, re-experiencing the traumatic event nist dapiprazole, which can block the effect of the sympathetic on (“flashbacks”), avoidance behavior (Marshall and Garakani, 2002; the iris when applied topically to the cornea (Yang et al., 2003; Stam, 2007). Apart from the reports by patients of an increased Hou et al., 2007), has been reported to inhibit the effect of con- tendency to startle, there is also laboratory evidence of sensi- ditioned fear on pupil diameter without affecting its effect on the tization of the startle reflex in PTSD (Butler et al., 1990; Orr light reflex response (Giakoumaki et al., 2005). et al., 1995; Morgan et al., 1996). However, it should be noted that exaggerated startle responses have not been reported in every CONCLUSIONS AND CLINICAL IMPLICATIONS study in which PTSD patients were compared with healthy con- The two physiological reflexes, acoustic startle reflex and pupil- trols (Grillon et al., 1996; Siegelaar et al., 2006). In addition to lary light reflex, considered in this review, are both sensitive to the enhancement of the muscular startle response, as recorded pain. The acoustic startle reflex, a somatic reflex, is enhanced by by EMG, there is also evidence of increased autonomic star- painful stimulation, whereas the pupillary light reflex, an auto- tle reactivity, as measured by heart rate and skin conductance nomic reflex, is inhibited by it. Although the two reflexes have responses, in PTSD (Orr et al., 1995; Shalev et al., 2000; Orr et al., very different mechanisms and underlying neuronal circuitries, 2003; Elsesser et al., 2004; Siegelaar et al., 2006). Interestingly, they are both under modulation by the LC, which itself is sen- the startle reactivity of PTSD patients has been shown to decline sitive to noxious stimulation, and via its widespread projections as symptoms subside in the course of therapy (Griffin et al., influences many somatic (e.g., muscle tone) and autonomic (e.g., 2012). The augmented startle reactivity in PTSD patients may pupillary activity) functions. be related to increased LC activity. It is generally recognized that Directly applied painful stimuli enhance the acoustic star- stress leads to the activation of the LC (Palkovits et al., 1995; tle response (“sensitization”) by augmenting the facilitation of Pacák and Palkovits, 2001; Valentino and Van Bockstaele, 2008). motoneurone activity by the LC. Noxious stimulation leads to Furthermore, the clinical presentation of patients with PTSD is pupil dilation (“reflex dilation”), which seems to be mediated by often complicated by pain and/or fear/anxiety, variables known different mechanisms in diurnal and nocturnal animals. In diur- to lead to the activation of the LC (see sections “Modulation nal animals, reflex dilation is likely to be due to the activation of of Pain by the LC,” “Effect of Pain: Sensitization,” and “Effect sympathetic premotor neurones in the LC, whereas in nocturnal of conditioned fear: fear-potentiation,” above). Finally, there is animals, it seems to be caused by the inhibition of parasympa- evidence of enhanced noradrenergic activity in patients suffer- thetic premotor neurones in the LC. Directly applied pain has no ing from PTSD (Southwick et al., 1993; Jacobsen et al., 2001). effect on the pupillary light reflex in man (it has not been studied Chronic pain has also been reported to be associated with exagger- in animals). ated startle responses (Carleton et al., 2006; Bakker et al., 2010). Painful stimuli applied indirectly via fear-conditioning, when Again, this observation may be related to enhanced LC activity, a neutral stimulus alone can evoke a state of anticipatory since pain is known to lead to the activation of the LC (see sec- fear/anxiety following its prior association with an aversive tion “Modulation of Pain by the LC,” above). Furthermore, the (painful) stimulus, also enhance the acoustic startle reflex (“fear- indirect activation of the LC by pain, via the generation of fear potentiated startle”), but they inhibit the pupillary light reflex and anxiety through the amygdala (see sections “Effect of Pain: (“fear-inhibited light reflex”). Fear-conditioning is mediated by Sensitization” and “Effect of conditioned fear: fear-potentiation,” the amygdala which has an excitatory influence on the LC. above), may also play a role in the enhancement of the startle

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response in chronic pain conditions. Finally, anxiety states have documented (for reviews, see Millan, 2003; Samuels and Szabadi, been reported to lead to alterations of the two physiological 2008b). reflexes. Both enhanced startle reflexes (Bakker et al., 2009; Reeb- In conclusion, the enhancement of the startle response in Sutherland et al., 2009) and diminished pupillary light reflexes PTSD, chronic pain conditions and anxiety states, and the atten- (Bakes et al., 1990) have been described in patients suffering uation of the pupillary light reflex response in anxiety disorder, from anxiety disorders. These observations are consistent with can be interpreted on the basis of the involvement of the LC in increased LC activity: the activation of the LC by anxiety is well the neuronal circuits controlling these two reflexes.

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