Standards in Pupillography.

The number of research groups studying the pupil is increasing, as is the number of publications. Consequently, new standards in pupillography are needed to formalize the methodology including recording conditions, stimulus characteristics, as well as suitable parameters of evaluation. Since the description of intrinsically photosensitive retinal ganglion cells (ipRGCs) there has been an increased interest and broader application of pupillography in ophthalmology as well as other fields including psychology and chronobiology. Color pupillography plays an important role not only in research but also in clinical observational and therapy studies like gene therapy of hereditary retinal degenerations and psychopathology. Stimuli can vary in size, brightness, duration, and wavelength. Stimulus paradigms determine whether rhodopsin-driven rod responses, opsin-driven cone responses, or melanopsin-driven ipRGC responses are primarily elicited. Background illumination, adaptation state, and instruction for the participants will furthermore influence the results. This standard recommends a minimum set of variables to be used for pupillography and specified in the publication methodologies. Initiated at the 32nd International Pupil Colloquium 2017 in Morges, Switzerland, the aim of this manuscript is to outline standards in pupillography based on current knowledge and experience of pupil experts in order to achieve greater comparability of pupillographic studies. Such standards will particularly facilitate the proper application of pupillography by researchers new to the field. First we describe general standards, followed by specific suggestions concerning the demands of different targets of pupil research: the afferent and efferent reflex arc, pharmacology, psychology, sleepiness-related research and animal studies.

Functional Organization of the Sympathetic Pathways Controlling the Pupil: Light-Inhibited and Light-Stimulated Pathways.

Pupil dilation is mediated by a sympathetic output acting in opposition to parasympathetically mediated pupil constriction. While light stimulates the parasympathetic output, giving rise to the light reflex, it can both inhibit and stimulate the sympathetic output. Light-inhibited sympathetic pathways originate in retina-receptive neurones of the pretectum and the suprachiasmatic nucleus (SCN): by attenuating sympathetic activity, they allow unimpeded operation of the light reflex. Light stimulates the noradrenergic and serotonergic pathways. The hub of the noradrenergic pathway is the locus coeruleus (LC) containing both excitatory sympathetic premotor neurones (SympPN) projecting to preganglionic neurones in the spinal cord, and inhibitory parasympathetic premotor neurones (ParaPN) projecting to preganglionic neurones in the Edinger-Westphal nucleus (EWN). SympPN receive inputs from the SCN via the dorsomedial hypothalamus, orexinergic neurones of the latero-posterior hypothalamus, wake- and sleep-promoting neurones of the hypothalamus and brain stem, nociceptive collaterals of the spinothalamic tract, whereas ParaPN receive inputs from the amygdala, sleep/arousal network, nociceptive spinothalamic collaterals. The activity of LC neurones is regulated by inhibitory α2-adrenoceptors. There is a species difference in the function of the preautonomic LC. In diurnal animals, the α2-adrenoceptor agonist clonidine stimulates mainly autoreceptors on SymPN, causing miosis, whereas in nocturnal animals it stimulates postsynaptic α2-arenoceptors in the EWN, causing mydriasis. Noxious stimulation activates SympPN in diurnal animals and ParaPN in nocturnal animals, leading to pupil dilation via sympathoexcitation and parasympathetic inhibition, respectively. These differences may be attributed to increased activity of excitatory LC neurones due to stimulation by light in diurnal animals. This may also underlie the wake-promoting effect of light in diurnal animals, in contrast to its sleep-promoting effect in nocturnal species. The hub of the serotonergic pathway is the dorsal raphe nucleus that is light-sensitive, both directly and indirectly (via an orexinergic input). The light-stimulated pathways mediate a latent mydriatic effect of light on the pupil that can be unmasked by drugs that either inhibit or stimulate SympPN in these pathways. The noradrenergic pathway has widespread connections to neural networks controlling a variety of functions, such as sleep/arousal, pain, and fear/anxiety. Many physiological and psychological variables modulate pupil function via this pathway.

GHB for cataplexy: Possible mode of action.

The sleep disorder narcolepsy is caused by the loss of orexinergic neurones in the lateral hypothalamus. A troublesome symptom of narcolepsy is cataplexy, the sudden loss of muscle tone in response to strong emotions. It can be alleviated by antidepressants and sodium oxybate (γ-hydroxybutyric acid (GHB)). It is likely that the noradrenergic nucleus locus coeruleus (LC) is involved since it is essential for the maintenance of muscle tone, and ceases to fire during cataplectic attacks. Furthermore, alpha-2 adrenoceptors proliferate in the LC in cataplexy, probably due to 'heterologous denervation supersensitivity' resulting from the loss/weakening of the orexinergic input to the LC. This would lead to the sensitization of the autoinhibition mechanism of LC neurones mediated by inhibitory alpha-2 adrenoceptors ('autoreceptors'). Thus the excitatory input from the amygdala to the LC, activated by an emotional stimulus, would lead to the 'switching off' of LC activity via the supersensitive auto-inhibition mechanism. GHB is an agonist at both γ-aminobutyric acid (GABA) GABA (B) and GHB receptors that may be a subtype of an extrasynaptic GABA(A) receptor. GHB may prevent a cataplectic attack by dampening the tone of LC neurones via the stimulation of inhibitory extrasynaptic GABA receptors in the LC, and thus increasing the threshold for autoinhibition.

Selective targets for arousal-modifying drugs: implications for the treatment of sleep disorders.

The level of arousal reflects the interaction between wakefulness-promoting and sleep-promoting nuclei located in the hypothalamus and brainstem. The nuclei and their connections constitute the sleep-arousal network. Mapping out this network, together with the neurotransmitters involved, has created a unique opportunity for the design of drugs for sleep disorders-it has become possible to target specific sites within the network with predictable effects on the level of arousal. Recent examples of this approach are orexin receptor and 5HT2A serotonin receptor antagonists and melatonin receptor agonists for the treatment of insomnia, and H3 histamine receptor antagonists for the treatment of excessive daytime sleepiness.

Functional neuroanatomy of the central noradrenergic system.

The central noradrenergic neurone, like the peripheral sympathetic neurone, is characterized by a diffusely arborizing terminal axonal network. The central neurones aggregate in distinct brainstem nuclei, of which the locus coeruleus (LC) is the most prominent. LC neurones project widely to most areas of the neuraxis, where they mediate dual effects: neuronal excitation by α1-adrenoceptors and inhibition by α2-adrenoceptors. The LC plays an important role in physiological regulatory networks. In the sleep/arousal network the LC promotes wakefulness, via excitatory projections to the cerebral cortex and other wakefulness-promoting nuclei, and inhibitory projections to sleep-promoting nuclei. The LC, together with other pontine noradrenergic nuclei, modulates autonomic functions by excitatory projections to preganglionic sympathetic, and inhibitory projections to preganglionic parasympathetic neurones. The LC also modulates the acute effects of light on physiological functions ('photomodulation'): stimulation of arousal and sympathetic activity by light via the LC opposes the inhibitory effects of light mediated by the ventrolateral preoptic nucleus on arousal and by the paraventricular nucleus on sympathetic activity. Photostimulation of arousal by light via the LC may enable diurnal animals to function during daytime. LC neurones degenerate early and progressively in Parkinson's disease and Alzheimer's disease, leading to cognitive impairment, depression and sleep disturbance.

Association of a deficit of arousal with fatigue in multiple sclerosis: effect of modafinil.

Multiple sclerosis (MS) is a multifocal demyelinating disease of the central nervous system, leading to chronic disability. Fatigue is a common and distressing symptom of MS which is unrelated to its clinical form, stage of development, the degree of disability, or the lesion load on magnetic resonance imaging. Fatigue in MS is associated with excessive daytime sleepiness and autonomic dysfunction. Recently it has been reported that the wakefulness-promoting drug modafinil may relieve fatigue in MS patients and ameliorate the associated cognitive difficulties. However, it is not clear to what extent the anti-fatigue effect of modafinil may be related to its alerting and sympathetic activating effects. We addressed this question by comparing three groups of subjects, MS patients with fatigue, MS patients without fatigue and healthy controls, matched for age and sex, on measures of alertness (self-ratings on the Epworth and Stanford Sleepiness Scales and on a battery of visual analogue scales; critical flicker fusion frequency; Pupillographic Sleepiness Test; choice reaction time) and autonomic function (systolic and diastolic blood pressure, heart rate, pupil diameter), and by examining the effect of a single dose (200 mg) of modafinil on these measures. MS patients with fatigue, compared with healthy controls, had reduced level of alertness on all the tests used; MS patients without fatigue did not differ from healthy controls. MS patients with fatigue had a reduced level of cardiovascular sympathetic activation compared to the other two groups. Modafinil displayed alerting and sympathomimetic effects in all three groups of subjects. As fatigue in MS is associated with reduced levels of alertness and sympathetic activity, modafinil may exert its anti-fatigue effect in MS by correcting these deficiencies. The anti-fatigue effect of modafinil may reflect the activation of the noradrenergic locus coeruleus (LC), since there is evidence that this wakefulness-promoting nucleus is damaged in MS, and that modafinil, probably via the dopaminergic system, can stimulate the LC. This article is part of a Special Issue entitled 'Cognitive Enhancers'.

Modulation of physiological reflexes by pain: role of the locus coeruleus.

The locus coeruleus (LC) is activated by noxious stimuli, and this activation leads to inhibition of perceived pain. As two physiological reflexes, the acoustic startle reflex and the pupillary light reflex, are sensitive to noxious stimuli, this review considers evidence that this sensitivity, at least to some extent, is mediated by the LC. The acoustic startle reflex, contraction of a large body of skeletal muscles in response to a sudden loud acoustic stimulus, can be enhanced by both directly ("sensitization") and indirectly ("fear conditioning") applied noxious stimuli. Fear-conditioning involves the association of a noxious (unconditioned) stimulus with a neutral (conditioned) stimulus (e.g., light), leading to the ability of the conditioned stimulus to evoke the "pain response". The enhancement of the startle response by conditioned fear ("fear-potentiated startle") involves the activation of the amygdala. The LC may also be involved in both sensitization and fear potentiation: pain signals activate the LC both directly and indirectly via the amygdala, which results in enhanced motoneurone activity, leading to an enhanced 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.

Comparison of pramipexole with and without domperidone co-administration on alertness, autonomic, and endocrine functions in healthy volunteers.

AIMS: To investigate the effects of the D2-receptor agonist pramipexole with and without the co-administration of the peripherally acting D2-receptor antagonist domperidone on measures of alertness, autonomic and endocrine function. METHODS: Sixteen male volunteers participated in four weekly sessions of pramipexole 0.5 mg, domperidone 40 mg, their combination, and placebo administered according to a balanced, double-blind design. Alertness (visual analogue scales (VAS), critical flicker fusion frequency, pupillographic sleepiness test), autonomic (pupil diameter, light and darkness reflexes, blood pressure, heart rate, salivation, temperature) and endocrine (prolactin, thyroid-stimulating hormone (TSH), growth hormone (GH)) functions were assessed. Data were analyzed with anova with multiple comparisons. RESULTS: The pre-post treatment changes in VAS alertness were reduced by pramipexole with and without domperidone (mean difference from placebo (95% confidence interval), mm): pramipexole -15.75 (-23.38, -8.13), combination -11.84 (-20.77, -2.91). Treatment condition significantly affected pupil diameter measured in different ways (resting pupil diameter (F(3,45) = 8.39, P < 0.001), initial diameter of the light reflex response (F(3,42) = 3.78, P < 0.05), and light (F(3,45) = 5.21, P < 0.005) and dark (F(3,45) = 3.36, P < 0.05) diameters of the darkness reflex response). Pramipexole without domperidone consistently increased pupil diameter on all measures (P < 0.05), whereas with domperidone only the increase in resting and dark diameters reached significance. Pramipexole reduced light reflex amplitude and increased latency, whereas the combination affected latency only. Concentrations of prolactin and TSH were increased by domperidone. Pramipexole reduced prolactin and increased GH concentrations. CONCLUSIONS: The attenuation of the central pupillary effects of pramipexole by domperidone indicates that domperidone had access to some central D2-receptors.

Modulation of the acoustic startle response by the level of arousal: comparison of clonidine and modafinil in healthy volunteers.

A sudden loud sound evokes an electromyographic (EMG) response from the orbicularis oculi muscle in humans together with an auditory evoked potential (AEP) and an increase in skin conductance (SC). Startle responses are inhibited by weak prepulses (prepulse inhibition, (PPI)) and may also be modified by the level of alertness. We compared the sedative drug clonidine and the alerting drug modafinil on sound-evoked EMG, AEP, and SC responses, on the PPI of these responses and on level of arousal and autonomic functions. Sixteen healthy male volunteers participated in four weekly sessions (clonidine 0.2 mg, modafinil 400 mg, their combination, placebo) in a double-blind, cross-over, balanced design. Responses were evoked by sound pulses of 115 and 85 dB (PPI) for 40 ms and recorded conventionally. Level of alertness, autonomic functions (pupil diameter, blood pressure, heart rate, salivation, temperature) and the plasma levels of the hormones prolactin, thyroid-stimulating hormone and growth hormone were also measured. Data were analyzed with analysis of variance with multiple comparisons. Both prepulses and clonidine attenuated all three startle responses and modafinil antagonized clonidine's effects on the EMG and AEP responses. None of the drugs affected PPI. Clonidine showed sedative and sympatholytic effects, and modafinil showed alerting and sympathomimetic effects. In conclusion, startle responses were susceptible not only to PPI but also to the level of arousal.

Relationship between sedation and pupillary function: comparison of diazepam and diphenhydramine.

AIMS: To examine the relationship between sedation and pupillary function by comparing the effects of diazepam and diphenhydramine on arousal and pupillary activity. METHODS: Fifteen male volunteers participated in three weekly sessions in which they received (i) diazepam 10 mg, (ii) diphenhydramine 75 mg and (iii) placebo, according to a balanced, double-blind protocol. Pupil diameter was measured with infrared pupillometry under four luminance levels. Alertness was assessed by visual analogue scales (VAS) and by critical flicker fusion frequency (CFFF). Blood pressure, heart rate and skin conductance were recorded by conventional methods. Data were analysed with analysis of variance (anova) with multiple comparisons. RESULTS: There were significant effects of ambient luminance (F3,42 = 305.7, P < 0.001) and treatment condition (F2,28 = 9.0, P < 0.01) on pupil diameter; diphenhydramine caused miosis at all luminance levels (P < 0.05). The light reflex response was not affected. Both active drugs reduced the pre-post treatment changes compared with placebo [mean difference from placebo (95% confidence interval)]: in CFFF (Hz), diazepam -0.73 (-1.63, 0.17), diphenhydramine -1.46 (-2.40, -0.52); and VAS alertness (mm), diazepam -11.49 (-19.19, -3.79), diphenhydramine -19.83 (-27.46, -12.20). There were significant effects of both session (F2,26 = 145.1, P < 0.001) and treatment (F2,26 = 5.5, P < 0.01) on skin conductance; skin conductance was reduced by both drugs (P < 0.05). CONCLUSIONS: The miosis by diphenhydramine and the reduction in skin conductance by both drugs may indicate central sympatholytic effects. A lack of a sympatholytic effect of diazepam on the pupil may be due to the masking of the miosis by mydriasis resulting from the inhibition of the parasympathetic output to the iris.

Drugs for sleep disorders: mechanisms and therapeutic prospects.

The past decade has witnessed an explosion of knowledge about the neural mechanisms that control sleep and arousal, triggered by two discoveries relating to the sleep disorder narcolepsy. Narcolepsy is caused by the loss of orexin-containing neurons in the hypothalamus, and a novel nonstimulant wakefulness-promoting drug, modafinil, alleviates excessive day-time sleepiness associated with the disorder. The level of arousal is controlled by an intricate interplay between distinct wakefulness- and sleep-promoting nuclei situated in the hypothalamus and brainstem and the interconnections between the nuclei and the neurotransmitters involved have been mapped. Wakefulness-promoting nuclei include the orexinergic lateral hypothalamic/perifornical area, the histaminergic tuberomammillary nucleus, the cholinergic pedunculopontine tegmental nucleus, the noradrenergic locus coeruleus, the 5-hydroxytryptaminergic raphe nuclei and possibly the dopaminergic ventral tegmental area. The major sleep-promoting nucleus is the GABAergic ventrolateral preoptic nucleus of the hypothalamus. Currently available and future drugs exert their therapeutic effects in the three major classes of sleep disorder (insomnia, hypersomnia, parasomnia) by modifying neurotransmission at distinct sites within the arousal-controlling neuronal network. This enables classification of therapeutic drugs for sleep disorders on the basis of their modes of action: drugs that interact with the GABAergic sleep-promoting system, drugs that interact with different wakefulness-promoting systems and drugs that modulate the level of arousal by mechanisms that do not initially involve the basic network (e.g. melatonin, adenosine). The development of novel therapeutic drugs for sleep disorders is based on the synthesis of molecular/cellular mechanisms and the sites of action within the arousal-controlling neuronal network.

Effects of 5-HT2A receptor stimulation on the discrimination of durations by rats.

We recently found that rats' ability to discriminate durations of exteroceptive stimuli is disrupted by the non-selective 5-HT receptor agonist quipazine. Ketanserin reversed this effect, suggesting that the effect may be mediated by 5-HT2A receptors. Here, we report that the 5-HT2A/2C receptor agonist 2,5-dimethoxy-4-iodoamphetamine (DOI) also disrupts temporal discrimination, and that this effect can be reversed by ketanserin and the highly selective 5-HT2A receptor antagonist (+/-)2,3-dimethoxyphenyl-1-[2-(4-piperidine)-methanol] (MDL-100907). Twenty rats were trained to discriminate durations in a discrete-trials psychophysical procedure. In each 50-s trial, a light was presented for t seconds, following which two levers (A and B) were presented. A response on A was reinforced if t < 25 s, and a response on B if t > 25 s. Logistic psychometric curves were fitted to the proportional choice of B (%B) for derivation of timing indices [T50: time corresponding to %B = 50; Weber fraction: (T75-T25)/2T50, where T75 and T25 are times corresponding to %B = 75 and 25, respectively]. DOI 0.25 mg kg (subcutaneous) significantly increased the Weber fraction and tended to increase T50. Ketanserin 2 mg kg (subcutaneous) did not alter either parameter, but completely antagonized the effects of DOI. Similarly, MDL-100907 0.5 and 1 mg kg (intraperitoneal) did not affect performance, but completely antagonized the effects of DOI. The results indicate that the mixed 5-HT2A/2C receptor agonist DOI disrupts temporal discrimination via stimulation of 5-HT2A receptors.