D. Warren Spence, M. A., Leonid Kayumov, Ph. D., Dabsm, Adam Chen, Ph. D., Alan Lowe, M. D., Umesh Jain, M. D., Martin A. Katzman, M. D., Jianhua Shen, M. D., Boris Perelman, Ph. D. and Colin M. Shapiro, mbbch, Ph. D

 Abstract

The response to acupuncture of 18 anxious adult subjects who complained of insomnia was assessed in an open prepost clinical trial study. Five weeks of acupuncture treatment was associated with a significant (p = 0.002) nocturnal increase in endogenous melatonin secretion (as measured in urine) and significant improvements in polysomnographic measures of sleep onset latency (p = 0.003), arousal index (p = 0.001), total sleep time (p = 0.001), and sleep efficiency (p = 0.002). Significant reductions in state (p = 0.049) and trait (p = 0.004) anxiety scores were also found. These objective findings are consistent with clinical reports of acupuncture's relaxant effects. Acupuncture treatment may be of value for some categories of anxious patients with insomnia.  Introduction

The incidence of insomnia is estimated to be 35% to 40% of the adult population. It currently affects more than 60 million Americans, and this figure is expected to grow to 100 million by the middle of the 21st century.^1,2 The defining characteristic of insomnia in the context of anxiety is a pattern of multiple arousals from sleep. Anxious subjects have difficulty maintaining sleep, spend less time in deep sleep, and their sleep is more fragmented than that of normal subjects.^3–5 Conversely, sleep deprivation may produce symptoms that fall within the total complex of anxiety.⁴ Although current opinion suggests that insomnia and anxiety are separate entities, their symptoms overlap considerably. Individuals with insomnia and individuals with anxiety have elevated psychosomatic profiles on psychological tests,^6–8 maintain chronically high states of arousal, and rely on an "internalizing" style of conflict resolution (DSM-IV, 4th Edition).⁹ These commonalities have prompted speculation¹⁰ that a common thread underlies the conditions, although their exact relationship, namely whether insomnia is the product of or simply a correlate of anxiety, is still inconsistently viewed in current diagnostic systems (i.e., the ICD-10, DSM-IV and ICSD [International Classification of Sleep Disorders]).¹¹ It is nevertheless true that the dual diagnostic pattern of anxious insomnia is the most commonly seen problem in sleep disorder clinics today.¹² The high rate of comorbidity between anxiety and insomnia, coupled with the high population incidence of insomnia, undoubtedly account for this phenomenon. Although we are unaware of any epidemiological studies on the incidence of anxiety which does not fulfill the criteria for a defined anxiety disorder, it is reasonable to infer that a large segment of the population may have "subsyndromal" anxiety, symptoms that are not associated with debilitating psychopathology, but which nevertheless produce a significant degree of mental discomfort.

Traditional treatment strategies for anxious insomnia have emphasized benzodiazepines. The useful anxiolytic effects of these agents have made them the most widely prescribed of all pharmaceuticals.¹³ The risks of benzodiazepines, however, are well documented and involve physical as well as psychological effects. These include their potential to promote dependence or acute toxicity in overdose.^14,15 Other adverse effects include sedation, psychomotor and cognitive impairment, memory loss, potentiation of other CNS depressants, and treatment-emergent depression.¹⁶ Acupuncture, which relies on the release of neurally active agents from endogenous stores, has been shown to have a superior side effect profile compared to some psychoactive drugs^17,18 and may thus represent a means for addressing the concerns about benzodiazepine therapy.

Evidence supporting acupuncture's utility as a treatment for insomnia has come from a variety of sources, including the non-western scientific literature. Among these, investigations by Nan and Qingming,¹⁹ Jiarong,²⁰ and Cangliang²¹ showed positive results. The shortcoming of these studies, however, is that their dependent measures have usually been inexact, relying mainly on subjective accounts of sleep experience or duration, and consequently, despite the consistency of their support for acupuncture, they are difficult to evaluate. Several European studies^22–24 used polysomnography to measure acupuncture effects on sleep disorders, but all failed to monitor nocturnal neurochemical changes which would have strengthened their experimental design.

It is known that stress mediation is multifactorial and strongly influenced by GABAergic²⁵ and dopaminergic neurotransmission.^26–27 The neurohormone melatonin may also be involved in these effects. Melatonin is a CNS depressant with anxiolytic,^28–29 mild hypnotic³⁰ and anticonvulsant actions³¹ which may be related to its enhancements of GABAergic^32–33 and striatal dopaminergic^34–35 transmission. The effect of melatonin on mood and chronobiological functions has been established in a number of studies. The pattern of melatonin secretion over a 24-hour period is widely accepted as a measure of circadian activity in humans.^36–37 This pattern is disrupted in insomnia. Compared to normal patients, those with insomnia have suppressed nocturnal outputs of melatonin^38–39 and are more likely to have histories of depression.⁴⁰ As noted above, the anxiolytic effects of melatonin have been recently established in rodent models.^41–43 In humans, abnormalities in melatonin secretion have been confirmed in patients with bipolar I disorder.⁴⁴ Taken together these findings support the inference that melatonin deficiency may play a key role in anxiety-associated insomnia.

Some evidence has also been provided that melatonin interacts with the opioid peptides.^45–46 Melatonin is both utilized and synthesized following acute pain episodes in humans,⁴⁷ the function of which may be to modulate fluctuations in opioid receptor expression and levels of beta-endorphin.⁴⁸ The relationship of melatonin with the opioidergic system is complex and not completely understood, although there is evidence that it has mixed opioid receptor agonist-antagonist activity.⁴⁹ In aggregate these findings lend support to the postulate of a "melatonin-opioid axis"⁴⁸ possibly serving a variety of protectant functions.

Evidence of the endogenous opioid basis of acupuncture analgesia has been supported both in human^50–51 and animal studies.^52–53 These have shown that acupuncture analgesia treatment increases CSF levels of met-enkephalin, beta-endorphin, and dynorphin and can be reversed by the opiate receptor blocker naloxone. These findings are relevant to the present study inasmuch as the opioids not only mediate analgesia they also play a central role in subjectively experienced stress. In normal human subjects plasma beta endorphin levels are increased just before or after a stressful experience,^54–55 and are associated with feelings of euphoria that is reported following, for example, bungee jumping.⁵⁶ In depressed patients elevated plasma beta endorphin levels are positively correlated with severe stress and phobia,⁵⁷ while anxious subjects show increases in beta endorphin immediately before and after cognitive and social stressors.⁵⁸ There is thus a reasonable basis for the inference that acupuncture modulates anxious responses and that these effects are mediated by the endogenous opioid system.

At the present time there have been only a few studies of acupuncture's effects on melatonin. In one of these however⁵⁹ acupuncture was found to promote increases in melatonin in the pineal, the hippocampus, and in serum in rats.

The study investigated the use of traditional (symptomatic) acupuncture without augmentation from herbs, pharmaceuticals or hormonal agents. Concentrations of a major melatonin metabolite 6-sulpha toxymelatonin (aMT6s) in urine were measured before and after the study (as described below). This was to evaluate changes in the neurohormone as released from endogenous sources (melatonin was not administered as an experimental treatment). For each subject the trial was conducted over a 7-week period during which the active phase of acupuncture therapy was 5 weeks (two sessions per week, 10 sessions in total). The acupuncture was administered by a master acupuncturist (AC) who was also the director of an acupuncture training program and clinic. The acupuncture needles were disposed of immediately after use and sterile technique was strictly observed. Each acupuncture session lasted approximately one hour. During the 1-week period preceding and following the active treatment phase, subjects were tested with polysomnography at an administratively convenient time in the Sleep Research Laboratory of the University Health Network, Toronto Western Hospital site. Figure 1 illustrates the design of the study.

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Figure 1.  Pre-post Experimental Design: Procedure and Chronology

Two consecutive overnight polysomnographic studies were performed at baseline (before treatment) and at the end of the 5 weeks of treatment with acupuncture. Polysomnographic results obtained on the first night during the before and after stages of the experiment were not included in the analysis to avoid a possible "first-night" effect.⁶¹ The sleep parameters included the sleep latency, sleep efficiency, the total sleep time, the arousal index, the percentage of REM sleep and REM latency, and the amount of time spent in stages 1 through 4. Additionally data were collected on the Alpha rating, an evaluative index of sleep quality⁶² which included an assessment of sleep fragmentation. For the baseline recordings, subjects chose their own retiring and wake up times as was consistent with their normal routine. Just before retiring on the second night of polysomnographic testing subjects were also asked to fill out several paper and pencil tests of mood and cognitive efficiency. These included the Toronto Alexithymia Scale,⁶³ a standard pre-sleep questionnaire; the Stanford Sleepiness Scale (SSS)⁶⁴; and a seven-item Fatigue Scale. Additionally they were asked to fill out the State-Trait Anxiety Inventory⁶⁵ to gauge the effect of acupuncture on anxiety. The Center for Epidemiological Studies Depression Scale (CES-D)⁶⁶ was used to assess the presence of depressive symptoms.

On the following morning, immediately after awakening, each subject completed a standard post-sleep questionnaire, the SSS, and the Fatigue Scale. Approximately 20 minutes after awakening, subjects assessed their level of fatigue and sleepiness using the following scales: the Fatigue Severity Scale, the Epworth Sleepiness Scale,⁶⁷ the Toronto Western Hospital Fatigue Questionnaire, the Fatigue Scale, and the FaST Adjective Checklist. The results from testing were consolidated to form a composite fatigue score (comfatigue), which has been validated in studies on patients with multiple sclerosis.⁶⁸

After completing the fatigue questionnaires, the subjects were asked to complete a complex verbal reasoning task.⁶⁹ Accuracy and time to complete the test were assessed.

During both the pre- and posttest assessment phases urine samples were collected and the concentration changes of aMT6s (which reflects the changes in endogenous levels of melatonin) were subsequently measured with a commercially available competitive immunoassay ELISA kit (Buhlmann Laboratories AG, Allschwil, Switzerland). At aMT6s concentrations 2.0 and 12.5 ng/ml the intraassay coefficients of variation were 5.5% and 3.5%; at concentrations 5.0 and 40.0 ng/ml the interassay coefficients of variation were 0.7% and 9.7%. As discussed above, the pattern of melatonin secretion has been widely accepted as a measure of circadian activity in humans,^36,37 and there is further evidence of decreases in melatonin output in patients suffering from insomnia. ^39,40,70

Statistical Analysis

The results of the polysomnographic recordings and psychometric testing were compared on a before and after basis for all subjects and are shown here as mean scores. The matched pairs t test was used to assess the statistical significance of these changes. The melatonin analysis was treated as a "two within-subjects variables experiment," a type of multiple repeated measures test, where the two within-subject factors were (a) "time of day" and (b) "phase of the experiment" (i.e., before or after the experiment). These comparisons were carried out using the Statistical Package for the Social Sciences software (SPSS for Windows). The null hypothesis was rejected if the differences were significant at the 5% level.

 Results

Sleep Duration and Sleep Quality Variables

The acupuncture treatment used in this study improved several polysomnographic parameters of sleep architecture. Among the sleep continuity variables, sleep onset latency (SOL) and the arousal index dropped significantly (p = 0.003 and p = 0.001, respectively), reflecting improvements in both sleep initiation and maintenance. The total sleep time (TST) and sleep efficiency similarly increased (p = 0.001 and p = 0.002, respectively). The Alpha index also improved significantly (p = 0.017). Some improvement in sleep quality was confirmed by the increase in the amount of time spent in stage three (slow wave) sleep (p = 0.023), but the amount of time spent in stage four sleep did not significantly change in the before-after comparison. The percentage of REM sleep and REM sleep latency, as well as the amount of time spent in stages one and two sleep remained unchanged following acupuncture.

Subjective Variables: Psychological Factors, Sleepiness, Fatigue, and Alertness

As shown in Table 2, both state and trait anxiety scores significantly improved (p = 0.049 and p = 0.004, respectively) following acupuncture. Additionally, scores on the CES-D showed significant improvements (p = 0.001). Scores on the Alexithymia Scale did not change significantly.

Scores on the Stanford Sleepiness Scale (SSS) indicated no significant differences (in the before and after comparison) when the test was administered just before the second night of sleep, but did show significant improvements (p = 0.019) when subjects were asked to report on their subjective sleepiness in the morning after the second night of sleep. The Fatigue Scale scores revealed a somewhat similar profile, with scores before sleep not showing any significant differences, but scores on the following morning indicated a significant improvement (p = 0.045) after 5 weeks of acupuncture. The improvement in fatigue scores were not paralleled by increases in alertness however: the ZOGIM-A, a test which measures alertness, indicated that the subjects felt significantly (p = 0.004) less alert following acupuncture. The composite fatigue scores (comfatigue) did not indicate any significant change. The timed test of cognitive skill indicated that subjects were able to perform the test more quickly (p = 0.001) following acupuncture, but the performance accuracy, while showing a small improvement, was not statistically significant.

6-Sulphatoxymelatonin Analysis

Urine analysis showed that nocturnal physiological levels of aMT6s increased following acupuncture and decreased during the morning and early afternoon (Figure 2).

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Figure 2.  Urinary concentrations of the melatonin metabolite 6-sulphatoxymelatonin (aMT6s) over a 24 hour cycle: comparison of baseline vs. post acupuncture treatment. The line graph indicates a statistically significant elevation (p = .002) of the metabolite in urine at night (Midnight-8am) and a significant decrease (p = .037) during the day (8am–3pm).

Analysis of the main effects showed a significant (p = 0.001) interaction between the two variables "time of day" (representing the four measurement periods 9 p.m. to midnight; midnight to 8 a.m.; 8 a.m. to 3 p.m. and 3 p.m. to 9 p.m.) and "phase of the experiment" (before versus after acupuncture), thus supporting the validity of individual time period comparisons on a pre- and post-treatment basis. No detectable changes (in urinary concentrations of aMT6s) were found for pairwise comparisons of periods 1 and 4 (9 p.m. to midnight, and 3 p.m. to 9 p.m.). Differences for periods 2 (midnight to 8 a.m.) and 3 (8 a.m. to 3 p.m.) however were significant (p = 0.002 and p = 0.037) reflecting postacupuncture increases in melatonin production at night and decreases during the morning and afternoon.

Discussion

Our initial hypotheses were confirmed by the results of the present investigation. In an open clinical trial of 18 subjects, the administration of 5 weeks of acupuncture, totaling ten treatment sessions, was associated with normalization in a 24-hour profile of urinary aMT6s and a number of objectively measured improvements in sleep continuity and sleep architecture. Additionally, significant improvements in self-reported fatigue and sleepiness paralleled these changes. The exception to this trend was the reduction in alertness as measured by the ZOGIM-A test. As discussed below the apparent inconsistency of reduced alertness following improvements in sleep quality may possibly have been the result of a transition into a more adaptive and qualitatively different type of alertness. Self assessed feelings of anxiety and depression decreased following acupuncture. These findings are fairly consistent with the results of previous investigations showing that acupuncture has a generalized anxiolytic effect,^71–73 and with other polysomnographic studies of acupuncture effects in insomnia.^22–24 The findings of nocturnal elevations in urinary aMT6s, indicating increased melatonin secretion, paralleled these changes. Melatonin regulates the rhythm of many functions and alterations in its secretory pattern have been found in a number of psychiatric disorders. These have included seasonal affective disorder, bipolar disorder, unipolar depression, bulimia, anorexia, schizophrenia, panic disorder, and obsessive-compulsive disorder,⁷⁴ but at present it has not been confirmed if these changes are causal to or simply a marker of other neurochemical dysfunctionalities. Further, it is not known if melatonin is equally involved in the development of the

pathophysiology of each of these disorders. Due to practical limitations we were able to investigate changes in only one neurally active agent, but clearly it would have been desirable to study acupuncture's effects on a range of neurotransmitters which are known to be closely linked to the etiology of anxiety or insomnia. Dysregulation of catecholamine secretion for instance has circadian variations which correlate closely with pathological anxiety states⁷⁵ and moreover have been shown to be regulated by melatonin injections.⁷⁶ Our findings thus raise intriguing questions about the nature and course of acupuncture effects at the neurochemical level. Studies are needed to further elucidate the role of norepinephrine as well as that of serotonin, dopamine, GABA in the changes we observed in melatonin secretion.

The results for the sleep architecture measurements showed no increases in the percentage of time spent in stages one or two, findings which have doubtful relevance for this clinical sample. Large improvements were seen however in the subjects' transition to stage three or slow wave sleep, reflective of significant gains in the quality of their sleep. A wide variability of responses in this observation reduced the significance level to p = 0.023. The percent of stage three sleep increased from a mean of 4.2% before treatment to a mean of 6.1% following treatment, closely approximating the normal mean of 7%. There was considerable variability in the amount of time spent in stage four sleep, with a number of subjects showing no improvement at all, thus accounting for the lack of statistical significance. The variability in responsiveness to acupuncture seen in, for instance, acupuncture analgesia treatment⁷⁷ has been known clinically and in scientific studies for some time. Although this variability has not been satisfactorily accounted for, one hypothesis is that psychological factors may be an impediment to treatment effectiveness. This is consistent with the findings of Widerstrom-Noga⁷⁸ and Creamer⁷⁹ showing that trait anxiety (measured by the STAI) can interfere with the effectiveness of acupuncture analgesia treatment. In this context our findings that, despite the variability of response, acupuncture improved overall sleep quality and had significant effects on anxiety are therefore noteworthy. The possibility that extreme scorers on trait anxiety are poor treatment candidates, or perhaps require additional treatment to show measurable changes, needs to be explored further with a sample that is larger than the one used in the present study.

In the present study subjects were screened to exclude those with clinical levels of psychopathology, including depression. Nevertheless a number of subjects showed elevated scores on the CES-D (depression) scale. This is in accordance with other findings showing that patients with insomnia may have symptoms of anxiety or depression which do not meet criteria for a specific mental disorder (DSM-IV, 4th Edition).⁹ In fact, symptom co-occurrence of anxiety and depression frequently exists in non-clinical samples which do not show serious sleep disturbance.⁸⁰ These symptoms were nevertheless reduced by acupuncture and are consistent with previous reports of acupuncture's effectiveness in treating mood disorders.^81–82

A finding that merits closer examination is the apparent lack of consistency implied in the failure of improvements in sleep quality to be accompanied by increasing alertness during the day. In our sample daytime alertness, as evaluated by self assessments or indirectly through measures of performance accuracy, either became worse or showed no improvement even though sleep quality was enhanced. Generally there is a positive correlation between tests of sleepiness (such as the Multiple Sleep Latency Test or MSLT) and daytime alertness (e.g., the Maintenance of Wakefulness Test, the MWT) (i.e., the better the nighttime sleep the greater the alertness during the day). In depressed patients however a negative relationship between the two tests is sometimes found.⁸³ Kayumov et al.⁸⁴ investigated this phenomenon in clinically depressed patients who also scored high on anxiety measures. In the depressed group the sleep latency on the MWT showed paradoxical increases (i.e., was reflective of alertness) in concordance with the severity of sleep disturbance, whereas in the non-depressed group this did not occur. Our own findings are consistent with these previous studies and support the view⁸⁴ that in depressed or anxious subjects the underlying factors which cause sleep disturbance will also produce heightened alertness during the day. This view emphasizes that qualitative differences exist in the "adaptive" alertness of non-anxious subjects, which is mobilized by relevant environmental stressors, and the accentuated or "vigilant" alertness of individuals suffering from excess emotional tension. In this group alertness is chronic and preferentially driven by internal rather than environmental demands, thus conferring to it a more invariant and non-discriminatory quality. Our finding therefore that alertness actually decreased following acupuncture may imply the substitution of one type of alertness for another rather than representing a decrement in cognitive efficiency. This possibility needs to be explored with testing instruments which are sensitive to these differences.

In this preliminary study acupuncture was shown to be of value as a therapeutic intervention for insomnia in anxious subjects and may therefore represent an alternative to pharmaceutical therapy for some categories of patients. Further, the central role attributed by classical and modern theories of personality to anxiety as the basis of most psychological defense mechanisms,^85,86 as well as the evidence that abnormalities in melatonin secretion are involved in a number of psychiatric conditions,⁷⁴ suggest that acupuncture may have broad applicability to other types of psychopathology in which quality of sleep is impaired. An important shortcoming of this study however was its lack of a control group with a placebo acupuncture condition. The findings therefore need to be confirmed with a study employing a more rigorous design.

1. 
   Hossain JL, Shapiro CM: The prevalence, cost implications, and management of sleep disorders: an overview. Sleep & Breathing 2002; 6:85–102
   
2. 
   Chilcott LA, Shapiro CM: The socioeconomic impact of insomnia. An overview. [Review] Pharmacoeconomics 1996; 10:Suppl 1:1–14
   
3. 
   Fuller KH, Waters WF, Binks PG, et al: Generalized anxiety and sleep architecture: a polysomnographic investigation. Sleep 1997; 20:370–376[Medline]
   
4. 
   Bourdet C, Goldenberg F: Insomnia in anxiety: sleep EEG changes. [Review] J Psychosom Res 1994; 38 Suppl 1:93–104
   
5. 
   Arraiga F, Paiva T: Clinical and EEG sleep changes in primary dysthymia and generalized anxiety: a comparison with normal controls. Neuropsychobiol 1990–91; 24:109–114
   
6. 
   London LH, Shulman B, Diamond S: The role of psychometric testing and psychological treatment in tension-type headache. Curr Pain Headache Repts. 2001; 5:467–471
   
7. 
   Stein MA, Mendelsohn J, Obermeyer, WH, et al: Sleep and behavior problems in school-aged children. Pediatrics 2001; 107:E60
   
8. 
   Norton GR, Norton PJ, Asmundson GJ, et al: Neurotic butterflies in my stomach: the role of anxiety, anxiety sensitivity and depression in functional gastrointestinal disorders. J Psychosom Res. 1999; 47:233–240
   
9. 
   American Psychiatric Association: Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition. Washington, D.C. American Psychiatric Association, 1994
   
10. 
    Morin CM: Insomnia, Psychological Assessment and Management. New York: Guilford Press, 1993
    
11. 
    Saletu B, Gruber G, Mandl M, et al: Clinical diagnosis in sleep laboratory patients based on ICD-10, DSM-III-R and ICSD classification criteria]. [German] Klinische Diagnosen bei Schlaflabor-Patienten basierend auf ICD-10-, DSM-III-R- und ICSD-Klassifikationskriterien. Wiener Medizin Wochen 1995; 145:656–662
    
12. 
    Ohayon MM, Caulet M, Lemoine P: Comorbidity of mental and insomnia disorders in the general population. Comp Psychiat 1998; 39:185–197[CrossRef] Baldessarini RJ: Drugs and the treatment of psychiatric disorders; psychosis and anxiety (Chapt. 18). In: Hardman JG, Limbird LE, Molinoff PB, Ruddon RW, Goodman Gilman A, The Pharmacological Basis of Therapeu`tics (ninth ed.). New York: McGraw-Hill, 1996
    
13. 
    Moller HJ: Effectiveness and safety of benzodiazepines. [Review] J Clin Psychopharm. 1999; 19(Suppl 2):2S-11S
    
14. 
    Lader MH: Limitations on the use of benzodiazepines in anxiety and insomnia: are they justified? European Neuropsychopharmacol 1999; 9(Suppl 6):S399-S405
    
15. 
    Nelson J, Chouinard G: Guidelines for the clinical use of benzodiazepines: pharmacokinetics, dependency, rebound and withdrawal. Canadian Society for Clinical Pharmacology. Can J Clin Pharmacol. 1999; 6:69–83
    
16. 
    Luo H, Meng F, Jia Y, et al: Clinical research on the therapeutic effect of the electro-acupuncture treatment in patients with depression. Psychiatry Clin Neurosci. 1998; 52 Suppl:S338-S340
    
17. 
    Han JS: Electroacupuncture: an alternative to antidepressants for treating affective diseases? Int J Neurosci 1986; 29:79–92[Medline]
    
18. 
    Nan L, Qingming Y: Insomnia treated by auricular pressing therapy. J Trad Chin Med 1990; 10:174–175[Medline]
    
19. 
    Jiarong L: Ten cases of somnambulism treated with combined acupuncture and medicinal herbs. J Trad Chin Med 1989; 9:174–175[Medline]
    
20. 
    Cangliang Y: Clinical observation of 62 cases of insomnia treated by auricular point imbedding therapy. J Trad Chin Med 1988; 8:190–192[Medline]
    
21. 
    Buguet A, Sartre M, LeKerneau J: Continuous nocturnal automassage of an acupuncture point modifies sleep in healthy subjects. Neurophysiologie Clinique 1995; 25:78–83[CrossRef][Medline]
    
22. 
    Montakab H, Langel G: The effect of acupuncture in the treatment of insomnia. Clinical study of subjective and objective evaluation. Schweizerische Medizinische Wochenschrift—Supplementum 1994; 62:49–54
    
23. 
    Dallakian IG, Vein AM, Kochetkov VD, et al: Relation between clinico-physiologic indices of sleep and the nature of stress and reflexotherapy application. Zhurnal Nevropatologii i Psikhiatrii Imeni S-S—Korsakova 1985; 85:534–539
    
24. 
    Biggio G, Concas A, Corda MG, et al: GABAergic and dopaminergic transmission in the rat cerebral cortex: effect of stress, anxiolytic and anxiogenic drugs. Pharmacology & Therapeutics. 48:121–42, 1990
    
25. 
    Labarca C, Schwarz J, Deshpande P, et al: Point mutant mice with hypersensitive alpha 4 nicotinic receptors show dopaminergic deficits and increased anxiety. Proceedings Nat Acad Sci 2001; 98:2786–2791[Abstract/Free Full Text]
    
26. 
    Glavin GB: Vulnerability to stress ulcerogenesis in rats differing in anxiety: a dopaminergic correlate. J Physiol, Paris 1993; 87:239–243[CrossRef][Medline]
    
27. 
    Nava F, Carta G: Melatonin reduces anxiety induced by lipopolysaccharide in the rat. Neurosci Lett 2001; 307:57–60[CrossRef][Medline]
    
28. 
    Arushanian EB, Chernysheva EM: The comparative effect of melatonin and diazepam on shifts in the anxiety-phobia status of rats evoked by damage to the amygdala. Eksperimentalnaia i Klinicheskaia Farmakologiia 1997; 60:7–9
    
29. 
    Kayumov L, Zhdanova IV, Shapiro CM: Melatonin, sleep, and circadian rhythm disorders. Sem Clin Neuropsychiat 2000a; 5:44–55
    
30. 
    Sandyk R: Melatonin and petit-mal epilepsy: an hypothesis. Int J Neurosci 1992; 65:83–90[Medline]
    
31. 
    Kopp C, Vogel E, Rettori MC, et al: Effects of melatonin on neophobic responses in different strains of mice. Pharmacol, Biochem Behav 1999; 63:521–6[CrossRef][Medline]
    
32. 
    Sanchez-Forte M, Moreno-Madrid F, Munoz-Hoyos A, et al: The effect of melatonin as anti-convulsant and neuron protector. Revista de Neurologia 1997; 25:1229–1234[Medline]
    
33. 
    Joo WS, Jin BK, Park CW, Maeng SH, Kim YS: Melatonin increases striatal dopaminergic function in 6-OHDA-lesioned rats. Neuroreport 1998; 9:4123–4126[Medline]
    
34. 
    Shieh KR, Chu YS, Pan JT: Circadian change of dopaminergic neuron activity: effects of constant light and melatonin. Neurorep 1997; 8(9–10):2283–2287
    
35. 
    Rosenthal NE: Plasma melatonin as a measure of the human clock (review). J Clin Endocrin Metab 1991; 73:225–226[Free Full Text]
    
36. 
    Lewy AJ, Sack RL: The dim light melatonin onset as a marker for circadian phase position. Chronobiol Int 1989; 6:93–102[Medline]
    
37. 
    Hajack G, Rodenbeck A, Staedt J, et al: Nocturnal plasma melatonin levels in patients suffering from chronic primary insomnia. J Pineal Res 1995; 19:116–122[Medline]
    
38. 
    Haimov I, Laudon M, Zisapel N, et al: Sleep disorders and melatonin rhythms in elderly people. BMJ 1994; 309:167[Free Full Text]
    
39. 
    Kripke DF, Elliot JA, Youngstedt SD, et al: Melatonin: marvel or marker? Ann Med 1998; 30:81–87[Medline]
    
40. 
    Kopp C, Vogel E, Rettori M, et al: Anxiolytic-like properties of melatonin receptor agonists in mice: involvement of mt1 and/or MT2 receptors in the regulation of emotional responsiveness. Neuropharmacol 2000; 39:1865–1871[CrossRef][Medline]
    
41. 
    Naranjo-Rodriguez EB, Osornio AO, Hernandez-Avitia E, Mendoza-Fernandez V, Escobar A: Anxiolytic-like actions of melatonin, 5-metoxytryptophol, 5-hydroxytryptophol and benzodiazepines on a conflict procedure. Prog Neuropsychopharmacol Biol Psychiat 2000; 24:117–129[CrossRef][Medline]
    
42. 
    Golombek DA, Pevet P, Cardinali DP: Melatonin effects on behavior: possible mediation by the central GABAergic system. Neurosci Biobehav Rev 1996; 20:403–412[CrossRef][Medline]
    
43. 
    Nurnberger JI, Adkins S, Lahiri DK, et al: Melatonin suppression by light in euthymic bipolar and unipolar patients. Arch Gen Psychiat, 2000; 57:572–579[Abstract/Free Full Text]
    
44. 
    Acuna-Castroviejo D, Escames G, Macias M, et al: Cell protective role of melatonin in the brain. [Review] J Pin Res 1995; 19:57–63
    
45. 
    Maestroni GJ, Conti A: Immuno-derived opioids as mediators of the immuno-enhancing and anti-stress action of melatonin. [Review] Acta Neurol 1991; 13(4):356–360.
    
46. 
    Nelson FA, Farr LA, Ebadi M: Salivary melatonin response to acute pain stimuli. J Pin Res 2001; 30:206–12[CrossRef]
    
47. 
    Barrett T. Kent S. Voudouris N: Does melatonin modulate beta-endorphin, corticosterone, and pain threshold? Life Sci 2000; 66:467–476[CrossRef][Medline]
    
48. 
    Ebadi M, Govitrapong P, Phansuwan-Pujito P, et al: Pineal opioid receptors and analgesic action of melatonin. J Pin Res 1998; 24:193–200[CrossRef]
    
49. 
    Ho WK, Wen HL: Opioid-like activity in the cerebrospinal fluid of pain patients treated by electroacupuncture. Neuropharmacol 1989; 28:961–6[CrossRef][Medline]
    
50. 
    Hardebo JE, Ekman R, Eriksson M: Low CSF met-enkephalin levels in cluster headache are elevated by acupuncture. Headache 1989; 8:494–497
    
51. 
    Han JS, Tang J, Ren MF, et al: Central neurotransmitters and acupuncture analgesia. Am J Chin Med 1980; 8:331–348
    
52. 
    Cheng RS, Pomeranz B: Electroacupuncture analgesia could be mediated by at least two pain-relieving mechanisms; endorphin and non-endorphin systems. Life Sci 1979; 25:1957–1962[CrossRef][Medline]
    
53. 
    Constantopoulos A, Papadaki-Papandreou U, Papaconstantinou E: Increased beta-endorphin but not Leu-enkephalin in plasma due to preoperative stress. Experientia 1995; 51:16–18[Medline]
    
54. 
    Schedlowski M, Fluge T, Richter S, et al: Beta-endorphin, but not substance-P, is increased by acute stress in humans. Psychoneuroendocrinol 1995; 20:103–110[CrossRef][Medline]
    
55. 
    Hennig J, Laschefski U, Opper C: Biopsychological changes after bungee jumping: beta-endorphin immunoreactivity as a mediator of euphoria? Neuropsychobiol 1994; 29:28–32[CrossRef]
    
56. 
    Darko DF, Risch SC, Gillin JC, et al: Association of beta-endorphin with specific clinical symptoms of depression. Am J Psychiatry 1992; 149:1162–1167[Abstract/Free Full Text]
    
57. 
    Gerra G, Zaimovic A, Zambelli U, et al: Neuroendocrine responses to psychological stress in adolescents with anxiety disorder. Neuropsychobiol 2000; 42:82–92
    
58. 
    Chao DM, Chen G, Cheng JS: Melatonin might be one possible medium of electroacupuncture anti-seizures. Acupunct Electro-Ther Res 2001; 26:39–48[Medline]
    
59. 
    Becker RE: Zung self-rating scale. In: Dictionary of behavioural Assessment techniques. (eds.) M Hersen and AS Bellak. Toronto, Pergammon Press. 1988
    
60. 
    Agnew H, Webb W, Williams R: The first night effect: an EEG study of sleep. Psychophysiology 1966; 2:263–266[Medline]
    
61. 
    Moldofsky H, Lue FA, Smythe HA: Alpha EEG sleep and morning symptoms in rheumatoid arthritis. Journal of Rheumatol 1983; 10:373–379
    
62. 
    Taylor GJ, Ryan DR, Bagby RM: Toward the development of a new self-report alexithymia scale. Psychother Psychosom 1985; 44:191–199[Medline]
    
63. 
    Hoddes E, Dement W, Zarcone V: The development and use of the Stanford Sleepiness Scale (SSS). Psychophysiology 1971; 9:150
    
64. 
    Spielberger CD (ed.) Anxiety: Current Trends in Theory and Research. New York, Academic Press. 1972
    
65. 
    Radloff LS: The CES-D scale: a self-report depression scale for research in the general population. Applied Psych Meas 1977; 1:385–401
    
66. 
    Johns MW: Reliability and factor analysis of the Epworth Sleepiness Scale. Sleep 1992; 15:376–381[Medline]
    
67. 
    Shapiro CM, Devins GM, Styra R, et al: Sleep features and fatigue in multiple sclerosis Sleep Res 1995; 24:387
    
68. 
    Baddeley AD: A three-minute reasoming test based on a grammatical transformation. Psychoneurol Sci 1968; 10:341–342
    
69. 
    Hajak G, Rodenbeck A, Staedt J, et al: Nocturnal plasma melatonin levels in patients suffering from chronic primary insomnia. J Pineal Res. 1995; 19:116–22
    
70. 
    Wang SM, Peloquin C, Kain ZN: The use of auricular acupuncture to reduce preoperative anxiety. Anesthes Analges 2001; 93:1178–1180[Abstract/Free Full Text]
    
71. 
    Eich H, Agelink MW, Lehmann E, et al: Acupuncture in patients with minor depressive episodes and generalized anxiety. Results of an experimental study. Fortschritte der Neurologie-Psychiatrie 2000; 68:137–144[CrossRef][Medline]
    
72. 
    Filshie J, Penn K, Ashley S, et al: Acupuncture for the relief of cancer-related breathlessness. Palliative Med 1996; 10:145–150[Abstract/Free Full Text]
    
73. 
    Pacchierotti C, Iapichino S, Bossini L, et al: Melatonin in psychiatric disorders: a review on the melatonin involvement in psychiatry. Frontiers Neuroendocrinol 2000; 22:18–32[CrossRef]
    
74. 
    McCann UD, Thorne D, Hall M, et al: The effects of L-dihydroxyphenylalanine on alertness and mood in alpha-methyl-para-tyrosine-treated healthy humans. Further evidence for the role of catecholamines in arousal and anxiety. Neuropsychopharmacol 1995; 13:41–52[CrossRef][Medline]
    
75. 
    Esquifino AI, Moreno ML, Steger RW: Effects of chronic melatonin administration on adrenal medulla catecholamine metabolism in adult male golden hamsters. J Pineal Res 1994; 16:154–158[CrossRef][Medline]
    
76. 
    Pomeranz B: Scientific basis of acupuncture (Chapt. 1). In: Stux G, Pomeranz B, eds. Acupuncture: Textbook and Atlas. New York: Springer Verlag, 1996
    
77. 
    Widerstrom-Noga E, Dyrehag LE, Borglum-Jensen L, et al: Pain threshold responses to two different modes of sensory Orofac Pain 1998; 12(1):27–34
    
78. 
    Creamer P, Singh BB, Hochberg MC, Berman BM: Are psychosocial factors related to response to acupuncture among patients with knee osteoarthritis?. Alt Ther Health Med 1999; 5(4):72–76
    
79. 
    Gottlib IH, Cane DB: Self-report assessment. In: Kendall PC, Watson D, eds. Anxiety and Depression: Distinctive and Overlapping Features. San Diego: Academic Press, 1989, pp 131–169
    
80. 
    Luo H, Meng F, Jia Y, et al: Clinical research on the therapeutic effect of the electro-acupuncture treatment in patients with depression. Psychiat Clin Neurosci 1998; 52 Suppl: S338–340
    
81. 
    Yang X, Liu X, Luo H, et al: Clinical observation on needling extrachannel points in treating mental depression. J Tradit Chin Med 1994; 14:14–18[Medline]
    
82. 
    Sangal RB, Thomas L, Mittler MM: Maintenance of wakefulness test and multiple sleep latency test. Measurement of different abilities in patients with sleep disorders. Chest 1992; 101:898–902[Abstract/Free Full Text]
    
83. 
    Kayumov L, Rotenberg V, Buttoo K, et al: Interrelationships between nocturnal sleep, daytime alertness, and sleepiness: two types of alertness proposed. Journal of Neuropsychiatry & Clinical Neurosciences 2000b; 12:86–90
    
84. 
    Freud A: The Ego and the Mechanisms of Defence. The Writings of Anna Freud Volume 2. (8 vols., Revised Edition, 5^th printing. First published 1936, in German) New York: International Universities Press, Inc., 1973
    
85. 
    Brenner C: Defense and defense mechanisms. Psychoanal Quart 1981; 50:557–569