Markus Kofler, MD, Innsbruck Medical University, Department of Neurology, Anichstrasse 35, A-6020 Innsbruck, Austria. Tel.: +43-512-504-23850; fax: +43-512-504-23852; e-mail: email@example.com
Hypocretin (orexin) deficiency plays a major role in the pathophysiology of narcolepsy–cataplexy. In animal models, hypocretinergic projections to the pedunculopontine nucleus are directly involved in muscle tone regulation mediating muscle atonia – a hallmark of cataplexy. We hypothesized that pedunculopontine nucleus function, tested with prepulse inhibition of the blink reflex, is altered in human narcolepsy–cataplexy. Twenty patients with narcolepsy–cataplexy and 20 healthy controls underwent a neurophysiological study of pedunculopontine nucleus function. Blink reflex, prepulse inhibition of the blink reflex and blink reflex excitability recovery were measured. Blink reflex characteristics (R1 latency and amplitude, and R2 and R2c latency and area under the curve) did not differ between patients and controls (P >0.05). Prepulse stimulation significantly increased R2 and R2c latencies and reduced R2 and R2c areas in patients and controls. However, the R2 and R2c area suppression was significantly less in patients than in controls (to 69.8 ± 14.4 and 74.9 ± 12.6%, respectively, versus 34.5 ± 28.6 and 43.3 ± 29.5%, respectively; each P <0.001). Blink reflex excitability recovery, as measured by paired-pulse stimulation, which is not mediated via the pedunculopontine nucleus, did not differ between patients and controls (P > 0.05). Our data showed that prepulse inhibition is reduced in narcolepsy–cataplexy, whereas unconditioned blink reflex and its excitability recovery are normal. Because the pedunculopontine nucleus is important for prepulse inhibition, these results suggest its functional involvement in narcolepsy–cataplexy.
Narcolepsy–cataplexy is a hypersomnia with a prevalence of approximately 0.05% in the general population. It is characterized by excessive daytime sleepiness, cataplexy, sleep paralysis, hypnagogic hallucinations and disturbed nocturnal sleep (Guilleminault et al., 2005). According to the revised International Classification of Sleep Disorders (ICSD-2), diagnosis of narcolepsy–cataplexy can be confirmed either by polysomnography or alternatively by assessing hypocretin-1 (orexin) levels in the cerebrospinal fluid (CSF). Current evidence suggests that narcolepsy–cataplexy is associated with a loss of at least 90% of hypocretinergic neurones located in the perifornical lateral hypothalamus (Nishino et al., 2000; Saper et al., 2001; Thannickal et al., 2000). Hypocretinergic neurones project to various areas in the central nervous system (CNS; De Lecea et al., 1998) such as, for example, amygdala (Guilleminault et al., 1998), medial medulla (Mileykovskiy et al., 2002) and pons (Peyron et al., 1998). In cats, hypocretinergic projections to the pedunculopontine nucleus (PPN) are involved directly in muscle tone regulation by mediating muscle atonia (Takakusaki et al., 2005). The authors speculated that cataplexy may thus result from both a decrease in the activity of the descending excitatory system via the mesencephalic locomotor region, as well as by an enhancement of the atonia-mediating system via the pedunculopontine/laterodorsal tegmental nuclei with sudden induction of muscle atonia by emotional signals generated in the limbic system (Takakusaki et al., 2005). Conversely, the PPN plays a central role in prepulse inhibition (PPI), which is a neurophysiological measure for sensorimotor gating and results in decreased responses of various reflexes, e.g. blink reflex and startle reaction (Valls-Soléet al., 1999, 2004). The presumed underlying circuits that mediate PPI of the startle reaction and the blink reflex have been reviewed recently by Valls-Solé (2012). A schematic illustration of the PPI circuit and its potential connection to the hypocretinergic system is illustrated in Fig. 1. Similarities of auditory and somatosensory prepulse effects at interstimulus intervals exceeding 100 ms on auditory and somatosensory blink reflexes (Valls-Soléet al., 1999) suggest that neuronal circuitry, which mediates PPI, is at least in part independent of stimulus modality. In this study, we investigated the blink reflex in a group of patients with narcolepsy and clear-cut cataplexy, which is highly predictive for hypocretin deficiency (Mignot et al., 2002), and in healthy controls. Based on the considerations above we hypothesized that PPI, which is mediated via the PPN, is altered in patients with narcolepsy–cataplexy, while the unconditioned blink reflex and its excitability recovery as measured by paired pulse stimulation, both of which are not relayed though the PPN, are unaffected in this disorder.
Subjects and methods
Selection of patients/controls
Patients > 18 years of age with narcolepsy and clear-cut cataplexy according to ICSD-2 criteria were included. Clear-cut cataplexy was defined according to Mignot et al. (1997) as brief episodes of weakness in the knees, jaw, face or neck triggered by laughter, joking, anger, elation, happiness or excitement with game-playing. Exclusion criteria were the use of any CNS-active medication other than modafinil 100–400 mg. Patients on modafinil were asked to skip the dosage on the day of the investigation. Patients with any definite or suspected diagnosis of comorbid psychiatric disease were not included in the present study. Healthy controls without a history of excessive daytime sleepiness, sudden onset of sleep or cataplexy, an Epworth Sleepiness Score > 10, an Ullanlinna Narcolepsy Scale score > 14 and without use of any CNS-active medication at the time of investigation were recruited from hospital staff or their relatives. This study was approved by the local ethical committee of Innsbruck Medical University in compliance with the Declaration of Helsinki. All participants granted written informed consent.
All participants were investigated at the Department of Neurology, Medical University Innsbruck, Austria, and at the Department of Neurology, Hochzirl Hospital, Zirl, Austria. We used routine electrodiagnostic equipment (Nicolet Viking IV, Madison, WI, USA). All neurophysiological studies were carried out in the supine position by the same investigator (M.K.), who ensured that neither patients nor controls fell asleep during the investigation. Before the experiments the state of alertness was assessed with the Stanford Sleepiness Scale. In addition, we controlled for the presence of emotional stimuli during the examination that could have triggered cataplexy.
Single sweeps of electromyographic activity of the orbicularis oculi muscles were recorded bilaterally with 10-mm-diameter surface gold electrodes attached to the skin, the active electrode in the middle portion of the muscle below each eye and the reference electrode lateral to the outer canthus. The electromyographic signal was amplified (× 1000) and band-pass filtered (30–3000 Hz). Blink reflexes were evoked by electrical stimuli (0.5-ms rectangular pulses) delivered to the right supraorbital nerve with surface electrodes, cathode over the supraorbital notch and anode 3 cm above on the forehead. We used a 10 × sensory threshold intensity to elicit the blink reflex in eight trials with at least a 10-s interval between two consecutive trials. Sensory threshold was defined as the minimal intensity that was perceived in at least four of eight stimulations. Care was taken to avoid measuring the R3 component (corresponding to the auditory startle reaction), which was occasionally present in the first or second response recording.
Prepulse inhibition of the blink reflex
The electrical stimulus used as a prepulse was delivered with ring electrodes to the digital nerves of the right index finger at twice the subject’s sensory threshold intensity, using constant current square wave electrical stimuli of 0.2 ms duration. In one healthy left-handed subject, the left index finger was used. The prepulse stimulus was applied 100 ms before the supraorbital nerve stimulus. Eight single sweeps were recorded with at least a 10-s interval between two consecutive trials.
Recovery curve of the blink reflex
For the excitability recovery curve of the R2 component of the blink reflex, paired pulses were delivered to the supraorbital nerve at the following interstimulus intervals: 160, 300 and 500 ms (Kimura and Harada, 1976; Kumru et al., 2010). Six traces were recorded and averaged online for each interstimulus interval, with at least a 15-s pause between each stimulus pair.
Data and statistical analysis
Following supraorbital nerve stimulation, we measured the latency and peak-to-peak amplitude of the early ipsilateral blink reflex component R1, and the latency and area under the curve (root mean square) of the ipsilateral (R2) and the contralateral (R2c) late blink reflex components. Eight responses on each side were averaged arithmetically for each subject separately. Group means of these ratios were then calculated for patient and control groups separately. For analysis of prepulse effects, we measured latency and peak-to-peak amplitude of R1 and latency and area under the curve of R2 and R2c following preceding finger stimulation. All eight responses on each side were averaged arithmetically for each subject separately. We also calculated the percentage change of the R2 and R2c areas in trials with a prepulse relative to trials without prepulse for each subject separately. Group means of these ratios were then calculated for patient and control groups separately. For analysis of blink reflex excitability, we measured the R2 area of six online averaged paired responses, and calculated the ratio of conditioned to unconditioned responses for both R2 and R2c separately, yielding the percentage recovery for each subject and each interstimulus interval tested. Means of these ratios were then plotted against the interstimulus intervals for patient and control groups separately in order to construct group mean blink reflex excitability recovery curves (Kimura and Harada, 1976; Kumru et al., 2010). Group results are expressed as mean ± standard deviation (SD). All statistical analyses were performed using spss version 18 (SPSS Inc., Chicago, IL, USA). Data distribution was tested using the Shapiro–Wilks W-test and analysis of variance (anova) for repeated measurements was performed to compare areas, latencies and amplitudes between test and conditioned trials and patients and controls. Post-hoc comparisons were performed using Student’s t-tests for independent or dependent samples with Bonferroni corrections. Sleep scores between patients and controls were compared using the Mann–Whitney U-test. Differences were considered significant at P < 0.05.
We studied 20 patients (14 males, six females) with a mean age of 45.1 ± 15.1 years (range 19–70 years). All patients were positive for human leucocyte antigen (HLA) DQB1*0602 and HLA DRB1*1501. Eighteen of the 20 patients were multiple sleep latency test (MSLT)-positive according to ICSD-2 criteria. Mean MSLT sleep latency was 2.7 ± 1.6. Mean number of sleep onset rapid eye movement (REM) periods was 3.3 ± 1.1. In the two MSLT-negative patients, CSF hypocretin-1 levels were not detectable. Twenty healthy volunteers (14 males, six females) with a mean age of 44.1 ± 13.7 years (range 19–74 years) served as control subjects. Patient and control demographics and sleepiness scores are given in Table 1. Sleepiness scores were significantly higher in patients compared to controls, while age and gender distribution did not differ significantly between patients and controls.
Table 1. Demographic data of patients and controls
SD, standard deviation; m, male; f, female; ESS, Epworth Sleepiness Scale; SSS, Stanford Sleepiness Scale; ULL, Ullan – linna Narcolepsy Scale; NS, not significant.
Age, mean ± SD
45.1 ± 15.1
44.1 ± 13.7
ESS, median (25th–75th percentile)
SSS, median (25th–75th percentile)
ULL, median (25th–75th percentile)
All participants tolerated this part of the study well. Blink reflex characteristics are listed in Table 2. Latency and amplitude of unconditioned R1, as well as latency and area under the curve of unconditioned R2 and R2c components did not differ between patients and controls.
Table 2. Neurophysiological results of unconditioned blink reflex (test) and prepulse inhibition (PPI) in patients with narcolepsy–cataplexy and in healthy control subjects
R1 latency (ms)
10.9 ± 1.0
10.7 ± 1.1
10.9 ± 1.0
10.7 ± 0.8
R1 amplitude (μV)
178 ± 179
221 ± 205
155 ± 116
232 ± 141
R2 latency (ms)
33.8 ± 3.8
39.3 ± 3.6
33.3 ± 3.9
36.4 ± 3.1
R2 area (μV × ms)
3845 ± 2396
1100 ± 985
4211 ± 1520
2723 ± 1302
R2c latency (ms)
34.8 ± 3.9
39.3 ± 3.7
33.8 ± 4.7
36.9 ± 3.5
R2c area (μV × ms)
3084 ± 2163
692 ± 554
3570 ± 1547
2311 ± 1664
Prepulse effect on the blink reflex in patients and controls
All participants completed the testing of PPI of the blink reflex. Examples of blink reflex responses without and with prepulse stimulation in a patient and a healthy control subject are shown in Fig. 2. Prepulse stimulation led to a significant increase in R1 amplitude (P < 0.01), without affecting R1 latency, in patients and controls. R2 and R2c latency increased significantly (P < 0.05) in both patients and controls, and the R2 and R2c areas were suppressed significantly by prepulse stimulation in both groups (P < 0.001). However, the amounts of R2 and R2c area suppression were significantly less in patients than in controls (P < 0.001, Fig. 3). R2 and R2c areas following prepulse stimulation were 30.1 ± 14.4% and 25.1 ± 12.6% in controls and 67.3 ± 25.4% and 61.7 ± 27.8% in patients, relative to respective baseline values. PPI investigation of the two patients with CSF-affirmed hypocretin-deficiency was similar to those of the remaining narcolepsy–cataplexy sample, although this comparison is clearly of limited value due to the small sample size. All other parameters were affected similarly by PPI in both groups. R2 and R2c area suppression by prepulse stimulation was not significantly different in patients with or without modafinil medication, and did not correlate with the Epworth Sleepiness Scale or Ullanlinna Narcolepsy Scale scores.
Recovery curve of the blink reflex
Sixteen patients and all 20 healthy controls underwent testing of blink reflex recovery. Of these, one patient refused to complete this part of the study; the data for two patients and one healthy control had to be excluded because of excessive tonic contraction of the orbicularis oculi muscles, which rendered exact area measurements impossible. Examples of blink reflex responses to paired stimuli in a patient and a healthy control subject are shown in Fig. 4. A repeated-measurements anova demonstrated significant effects of interstimulus interval on R2 (F = 113,4; P < 0.001) and R2c areas (F = 77,3; P < 0.001), but these effects did not differ between patients and controls (F = 0.04 for R2, F = 0.93 for R2c). In both patients and controls, suppression of the R2 and R2c components following the second stimulus decreased similarly with increasing interstimulus intervals (Fig. 5).
To our knowledge, this is the first study demonstrating that PPI is altered in narcolepsy–cataplexy compared to controls. In addition, for the first time in a non-extrapyramidal motor disorder, this study reveals that PPI of the blink reflex can be deficient in the absence of altered blink reflex excitability recovery. The results concur with functional involvement of the PPN without affecting the blink reflex circuitry.
Prepulse inhibition of the blink reflex is one method for the assessment of brainstem function. It permits determination of the percentage inhibition induced in the blink reflex by a preceding stimulus of the same or another modality (prepulse stimulus), which is so weak that it does not cause any response on its own. The functional anatomy of PPI is still understood incompletely. From animal experiments it is known that the PPN, nucleus reticularis pontis caudalis and substantia nigra pars reticulata are crucial structures involved in the PPI circuit (Fendt et al., 2001; Koch et al., 1993; Reese et al., 1995; Swerdlow et al., 2001). Furthermore, the PPN is the main structure within hierarchical circuits governing brainstem reflexes such as the blink reflex (Rossi and Scarpini, 1992; Valls-Soléet al., 1999) or the startle reaction (Ison et al., 1990; Kofler et al., 2006).
Based on the current animal model-driven hypothesis, that hypocretinergic projections to the PPN are involved directly in muscle tone regulation by mediating muscle atonia (Takakusaki et al., 2005), cataplexy may thus result from both a decrease in the activity of the descending excitatory system via the mesencephalic locomotor region as well as by an enhancement of the atonia-mediating system via the pedunculopontine/laterodorsal tegmental nuclei with sudden induction of muscle atonia by emotional signals generated in the limbic system (Fuller et al., 2007; Mileykovskiy et al., 2002; Takakusaki et al., 2005). Evidence of PPN involvement in human narcolepsy–cataplexy is extremely scarce, and based on anecdotal symptomatic narcolepsy cases due to structural lesions in the area of the PPN (see e.g. Mathis et al., 2007) or indirect assumptions from deep brain stimulation studies of the PPN in Parkinson’s disease (Arnulf et al., 2010) and small postmortem narcolepsy case studies (Aldrich et al., 1994; Mamelak, 1991). The finding of a functional involvement of the PPN in human narcolepsy–cataplexy is supported further by investigation of the auditory startle reaction in narcolepsy–cataplexy: Lammers et al. (2000) investigated 14 patients with narcolepsy–cataplexy compared to 12 healthy controls and demonstrated that auditory startle reaction is increased in narcolepsy–cataplexy, consistent with reduced inhibition from higher-order control levels such as the PPN.
In our study, blink reflex parameters did not differ between patients and controls. This is in line with existing literature showing, at least in the cataplexy-free state, no difference in blink reflex in narcolepsy–cataplexy compared to controls (Khatami et al., 2007; Marx et al., 1998). In addition, Khatami et al. (2007) found that patients with narcolepsy–cataplexy failed to exhibit startle potentiation during unpleasant stimuli, supporting the hypothesis of an amygdala dysfunction in human narcolepsy–cataplexy. In contrast to the cataplexy-free state, Quinto et al. (2005) reported a patient who presented with an enhanced blink reflex R2 component during status cataplecticus compared to the symptom-free interval, suggesting either hyperexcitability or disinhibition of brainstem interneurones during cataplexy.
Blink reflex circuit excitability can also be modulated in a paired pulse paradigm by presentation of two identical reflex-evoking stimuli. In healthy subjects, the response evoked by the second stimulus is smaller than the response evoked by the first stimulus with interstimulus intervals below 1 s. Blink reflex excitability recovery is altered in various neurological disorders, as reviewed recently by Valls-Solé (2012). To the best of our knowledge, there are no investigations on blink reflex recovery curves in narcolepsy or any other sleep disorders. We found no difference in blink reflex recovery between narcolepsy–cataplexy patients and healthy controls. The difference between the blink reflex recovery curve and PPI results could be explained by differences in the reflex circuitries of both reflexes. Whereas the PPN plays a major role in PPI it is bypassed in the blink reflex circuit, as tested by the paired pulse paradigm (Valls-Soléet al., 2004).
Of note, the blink reflex and its excitability recovery were reported to be affected by sleep (Kimura and Harada, 1976). Although the finding that unconditioned blink reflex parameters did not differ significantly in patients and controls in the present study favours the idea that PPI changes result from the disease itself, we cannot definitively exclude a contribution of excessive sleepiness in the patient group to PPI changes.
Hypocretin deficiency was affirmed in the CSF in only two patients, whereas in the majority of patients no affirmation of CSF hypocretin deficiency was performed. Although HLA DQB1*0602 positivity and clear-cut cataplexy are highly predictive for hypocretin deficiency (Mignot et al., 2002), such a deficiency can be assumed only for the total group. Nevertheless, PPI findings of the two affirmed hypocretin-deficient cases are similar to the remaining untested patient sample.
Half the patients with narcolepsy–cataplexy were on modafinil therapy. To minimize any potential influence, we asked patients to skip the modafinil dosage on the day of the examination, and found that PPI was not significantly different in patients with or without modafinil medication. This may, at least, indicate no sustained influence of modafinil on the brainstem reflexes tested. Whether acute modafinil, which is postulated to exert an anti- gamma-aminobutyric acid (GABA)ergic action (Joo et al., 2010; Nardone et al., 2010), could affect PPI of the blink reflex cannot be answered by the design of the present study. Notably, GABAergic modulation of brainstem reflexes by baclofen has been shown recently (Kumru et al., 2009, 2010).
In summary, our data show that PPI is clearly altered in narcolepsy–cataplexy, whereas unconditioned blink reflex and its excitability recovery are normal. As the PPN appears to play a crucial role in prepulse inhibition, these results suggest a functional involvement of the PPN in narcolepsy–cataplexy.
All authors have neither personal nor financial interests in the subject matter of the paper, nor are they involved with organizations with financial interest in the subject matter of the paper.