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Original Studies |
Departments of Psychiatry (M.V., J.D.B., G.R.H., D.S.C.), Child Psychiatry (G.M.A.), and Anesthesiology (T.M.H.), Yale University School of Medicine, New Haven, Connecticut 06519; and Department of Psychiatry and Behavioral Sciences, Emory University School of Medicine (M.J.O., C.B.N.), Atlanta, Georgia 30322
Address all correspondence and requests for reprints to: Meena Vythilingam, M.D., Mood and Anxiety Disorders Research Program, National Institute of Mental Health, 9000 Rockville Pike, Building 10, Room 4N222, Bethesda, Maryland 20892-1381. E-mail: Vythim{at}nih.gov
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Abstract |
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2-adrenergic antagonist) would have stimulatory action
on non-HPA CRH neurons, whereas naloxone (a µ-opioid receptor
antagonist) would not have this effect. Adult normal volunteers
received iv yohimbine (n = 5; 0.4 µg/kg), naloxone (n = 4;
125 µg/kg), or placebo (n = 3; 0.9% saline). Cerebrospinal
fluid (CSF) was collected continuously, and concentrations of CSF CRH,
CSF norepinephrine (NE), and plasma cortisol were measured.
Administration of either yohimbine or naloxone caused significant
increases in plasma cortisol concentrations over time. Although
yohimbine robustly increased CSF NE levels and appeared to increase CSF
CRH levels, these effects were not seen after naloxone or placebo
administration. Intraindividual correlations were not observed between
the measured concentrations of plasma cortisol and CSF CRH for any of
the subjects. The results support the idea that CSF CRH concentrations
reflect the activity of non-HPA CRH neurons. Although both yohimbine
and naloxone stimulated the HPA axis, only yohimbine appeared to have
stimulatory effects on central NE and non-HPA CRH. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The vast majority of hypothalamic CRH neurons are found in the paraventricular nucleus (PVN) and project to the median eminence, where CRH is released from nerve terminals into the primary plexus of the hypothalamo-hypophyseal portal system (9). The released CRH plays a major role in the stress response by stimulating the production and release of ACTH and a resultant increase in plasma cortisol concentrations. The CRH neurons of the PVN also project to areas other than the median eminence, including the brainstem and locus coeruleus (10). CRH perikarya are also located in regions outside the hypothalamus, including such diverse areas as the cerebral cortex, amygdala, bed nucleus of the stria terminalis, central gray area, dorsal tegmentum, locus coeruleus, parabrachial nucleus, dorsal vagal nucleus, and inferior olive (9, 11, 12). The extrahypothalamic CRH neurons and the hypothalamic neurons projecting to areas other than the median eminence will be collectively referred to as non-HPA CRH. The production of anxiety-like behavioral and autonomical effects after central administration of CRH in hypophysectomized animals suggests that the non-HPA CRH contributes to the nonendocrine aspects of the stress response syndrome (13). These data and related evidence have focused attention on the role of non-HPA CRH circuits in mediating behavioral, autonomic, and cognitive responses to stress.
Preclinical evidence has supported extensive interactions between CRH and the noradrenergic, opioidergic, and serotonergic systems (14, 15, 16, 17, 18). The interaction of norepinephrine (NE) and CRH is especially important, as these two neurotransmitters are considered the predominant central mediators of the stress response. Studies have shown that CRH serves as an excitatory neurotransmitter in the locus coeruleus, where it causes a dose-dependent increase in discharge rate, enhances NE release, and produces electroencephalographic activation in the cerebral cortex (10, 19, 20, 21, 22). However, the effect of NE on PVN CRH neurons projecting to the median eminence has been a controversial subject (18, 23, 24, 25, 26, 27). More recent preclinical studies support a stimulatory role for NE in the functioning of PVN CRH neurons (28, 29, 30). However, there is little information regarding possible noradrenergic regulation of CRH neurons located outside the hypothalamus.
Opioid neuropeptides are released during stress and tonically inhibit CRH release from the hypothalamus without having direct effects on either the pituitary or adrenal glands (31). Naloxone, a µ-opioid receptor antagonist, appears to have a direct stimulatory effect on PVN CRH neurons, resulting in elevated plasma ACTH and cortisol concentrations (32, 33, 34, 35). However, little is known about opioid regulation of non-HPA CRH neurons.
Studies in human subjects with mood and anxiety disorders have to a great extent relied upon cerebrospinal fluid (CSF) CRH concentrations as an index of central CRH function (6, 7, 36). However, lumbar CSF CRH levels do not appear to be derived principally from the hypothalamic-pituitary component of the CRH system. This is supported by the observation that the diurnal rhythm of CSF CRH in nonhuman primates and humans is not closely linked to that of plasma ACTH and cortisol concentrations (37, 38, 39, 40). Furthermore, pharmacological agents that either decrease or increase plasma ACTH and cortisol do not cause a corresponding change in CSF CRH concentrations or their diurnal variation (39). This is in contrast to the CSF concentrations of hormones such as LH-releasing hormone, which is closely linked to changes in pituitary LH secretion (41). Taken together, the data suggest that CSF CRH concentrations may reflect release from non-HPA CRH neurons.
Methods for serially sampling CSF in awake human subjects have been
developed (42, 43). This methodology when combined with
pharmacological challenges may provide a useful technique to evaluate
the regulation of the non-HPA CRH system in humans. Determination of
the regulatory influence of the noradrenergic and opioid systems on CSF
CRH in normal healthy subjects may furnish clues to the CSF CRH
elevations previously reported in patients with major depression and
posttraumatic stress disorder and may offer insight into the mechanisms
of treatment response in these disorders. With this in mind, we have
investigated possible noradrenergic and opioid regulation of CRH
neurons in healthy human subjects using the
2-adrenergic antagonist yohimbine and the
µ-opioid antagonist naloxone.
We have attempted to test the hypothesis that yohimbine has a stimulatory action on non-HPA CRH neurons. In contrast, naloxone was not expected to have this effect. More specifically, we hypothesized that iv administration of yohimbine would result in a significant increase in the non-HPA CRH (as reflected in CSF CRH) and in HPA functioning (as reflected in plasma cortisol), whereas naloxone was expected to increase HPA functioning without affecting non-HPA CRH.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Subjects received dextrose-0.9% normal saline at a rate of 100 mL/h 1 h before and during the study through an iv catheter placed in the forearm. Blood samples for measurement of plasma cortisol and catecholamine concentrations were obtained through the same catheter, with adequate precaution to avoid dilution with iv fluids. Blood samples were obtained at half-hourly intervals before administration of yohimbine, naloxone, or placebo and at more frequent intervals immediately after the injection at noon. A total volume of 180 mL blood was obtained during the course of the study. Blood was immediately placed on ice, and plasma was prepared within 1 h of sampling. Plasma was then stored at -70 C for subsequent analysis.
Behavioral measures included clinician and self-ratings of anxiety and mood states, obtained at predetermined time points before and during the course of the study. A trained research nurse clinician administered the Visual Analog Scales to measure baseline anxiety, nervousness, fear, irritability, drowsiness, and energy. The total score on the Patient-Rated Anxiety Scale (PRAS) (45) was also used to quantify the subject’s experience of anxiety.
The subarachanoid catheter was withdrawn at 1830 h, and subjects were allowed to resume eating regular meals. They were asked to elevate their heads gradually and were allowed to sit in bed and use the bathroom. The next morning, subjects were encouraged to ambulate, and iv fluid was discontinued at 1200 h if subjects did not develop adverse effects. Subjects were discharged that evening if they exhibited no side effects and were able to walk comfortably. A physician and a trained research nurse were present during the entire study. Nursing care was also available before and after the study. Subjects were followed for up to 2 weeks after the procedure or longer if indicated.
Side effects
Of the nine healthy subjects who received either yohimbine or naloxone, four developed severe or persistent headache 1–2 days after removal of the catheter. The headache resolved immediately after treatment with a blood patch placed by an anesthesiologist. None of the subjects who received placebo required a blood patch. Two subjects developed mild headaches that responded to iv hydration and orally administered analgesics. One subject developed mild low back pain that resolved with nonsteroidal analgesics, whereas two subjects had no side effects. It is unclear why rates of severe headache were substantially higher in these younger healthy subjects compared with the rates seen in older patients [Geracioti, T. D. (University of Cincinnati College of Medicine, Cincinnati, OH), personal communication]. It is possible that differences in the type, gauge, and angle of the bend of the needle in addition to body posture during and after the procedure contributed to the side effects (Halaszynski, T. M., et al., manuscript in preparation). In view of the high rate of headache after the continuous CSF collection procedure, enrollment in the study was suspended, and the samples of the completed subjects were analyzed.
Biochemical analysis
Concentrations of CSF CRH were measured in duplicate using a modification of a previously described specific RIA for CRH (46). Duplicate 450-µL aliquots of CSF were lyophilized, reconstituted in 200 µL assay buffer, and incubated at 4 C for 18 h with 100 µL antiserum (oC33) raised in rabbits against ovine CRH at a final dilution of 1:21,875 in assay buffer containing 1% normal rabbit serum. Radiolabeled [125I]Tyr0-rat/human CRH (20,000 cpm in 50 µL buffer) was then added to each tube. After incubation for 24 h at 4 C, 10 µL goat antirabbit serum were added to precipitate bound CRH. The standards containing 450 µL artificial CSF and 0.625–640 pg CRH/tube were prepared using rat/human CRH and were treated identically as the human CSF samples. The sensitivity of the assay was 1.25 pg/tube, with 50% displacement of radiolabeled CRH (IC50) at 30 pg/tube. The inter- and intraassay coefficients of variation have been measured every 6 months for the past 13 yr with a set of identical samples (pooled human CSF) in two separate assays. The difference between these values has ranged from 10–13% for interassay measurements and from 2–6% for intraassay measurements over this period of time. Plasma cortisol concentrations were determined using commercially available radiometric assay kits supplied by INCSTAR Corp. (Stillwater, MN); within-day and day-to-day coefficients of variation of 5–9% were observed.
Concentrations of plasma and CSF NE and other metabolites [homovanillic acid and 3-methoxy-4-hydroxyphenylglycol (MHPG)] were measured using high performance liquid chromatography (47).
Statistics
Concentrations of CSF CRH, CSF NE, and plasma cortisol were expressed as a percentage of the mean of three baseline values (-15, -45, and -75 min points). Two-way repeated measures ANOVA with time as the repeated measure was used to determine changes in CSF CRH, CSF NE, and plasma cortisol after yohimbine and naloxone administration. Effects of yohimbine, naloxone, and placebo were also examined using one-way repeated measures ANOVA. Area under the curve (AUC) responses above the baseline were calculated using the trapezoidal method and compared using the Mann-Whitney U test. Relationships between peak change in biological and behavioral measures were estimated using Spearman’s rank order correlation.
Mean arterial pressure was calculated using the formula: diastolic blood pressure + 1/3 (systolic blood pressure - diastolic blood pressure). Changes in behavioral measures, mean arterial pressure, and pulse were assessed using repeated measures ANOVA.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Drug and placebo effects on CSF CRH concentrations are shown in
Fig. 1
. A repeated measures ANOVA
comparing the CSF CRH responses to yohimbine, naloxone, and placebo did
not reveal a significant effect for drug condition (F = 0.85;
df = 2,9; P = 0.46). However, a significant effect
was observed for time (F = 3.08; df = 6,54; P
= 0.01), and the drug by time interaction approached statistical
significance (F = 1.58; df = 12,54; P =
0.12). Due to the small size of the groups being compared, a one-way
repeated measure ANOVA was also performed to examine the effects of
yohimbine on CSF CRH. In this analysis, CSF CRH increased
significantly over time after administration of yohimbine (F =
3.92; df = 14; P = 0.0001). In contrast, CSF CRH
concentrations did not change significantly after administration of
either naloxone (F = 0.98; df = 14; P =
0.49), or placebo (F = 0.33; df = 6; P =
0.91). The changes in CSF CRH after yohimbine, naloxone, and placebo
administration were also compared by examining the AUC responses.
Although the AUC responses (percentage of baseline multiplied by time
interval in hours; % B x h) for CSF CRH after yohimbine
administration (-43%, 39%, 263%, 562%, and 593% B x h)
tended to be higher than those seen for either naloxone (-49%, 17%,
89%, and 241% B x h) or placebo (-18%, 10%, and 161% B
x h), the differences between yohimbine and the other agents did not
achieve statistical significance (by Mann-Whitney U test; yohimbine
vs. placebo: z = 1.34; P = 0.18;
yohimbine vs. naloxone: a = 1.47; P =
0.14).
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Plasma cortisol increased significantly over time after
administration of both drugs (F = 6.59; df = 10,70;
P = 0.0018; see Fig. 2).
There was no significant main effect of drug (F = 0.15; df =
1,7; P = 0.71), nor was a significant drug by time
interaction observed (F = 1.17; df = 10,70; P
= 0.33). In addition, there was no significant difference between the
plasma cortisol AUC responses seen after administration of naloxone
(-24%, 73%, 133%, and 340% B x h) or yohimbine (69%, 116%,
174%, 175%, and 273% B x h; by Mann-Whitney U test: z =
0.74; P = 0.46). In general, the increases seen in
plasma cortisol after the administration of yohimbine were similar to
those observed after naloxone administration. Plasma cortisol responses
to yohimbine and naloxone administration could not be compared with the
placebo response due to limited sample availability.
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When the effects of yohimbine and naloxone on CSF NE
concentrations were examined, significant effects of drug (F =
52.8; df = 1,7; P = 0.0002) and time (F =
11.4; df = 14,98; P = 0.0001), as well as a
significant drug by time interaction (F = 7.42; df = 14,98;
P = 0.0001) were observed (see Fig. 3). CSF NE was significantly elevated
compared with baseline values at all time points from 1–5.5 h after
yohimbine administration (range of F values, 11.5–164; range of
P values, 0.02–0.03). Similarly, the CSF NE AUC responses
after yohimbine administration (104%, 224%, 238%, 243%, and 332%
B x h) were significantly greater than those after naloxone
(-133%, -98%, -39%, and 33% B x h; by Mann-Whitney U test:
z = 2.45; P = 0.01). The CSF NE response after
drug administration could not be compared with the placebo response due
to limited sample availability.
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There was no significant change in CSF homovanillic acid, plasma MHPG, or CSF MHPG concentrations over time after the administration of either yohimbine or naloxone (by two-way repeated measures ANOVA).
Relationships among CSF CRH, CSF NE, and plasma cortisol
There was no significant correlation between plasma cortisol AUC responses and CSF CRH AUC responses for individual patients; this was the case whether subjects who received yohimbine and naloxone were considered separately or together. None of the point by point intraindividual correlations calculated for plasma cortisol and CSF CRH concentrations observed from 0900–1800 h were significant. To account for the expected lag time for CSF CRH secreted in the brain to reach the CSF compartment, correlations were also performed between plasma cortisol levels at a specific time point and CSF CRH concentrations observed 1, 2, and 3 h after that corresponding time. None of the lagged correlations between plasma cortisol and CSF CRH were significant. The CSF CRH AUC and CSF NE AUC responses obtained for each patient were not correlated. This was the case when all patients were pooled and when yohimbine and naloxone patients were considered separately. In addition, none of the intraindividual correlations calculated for CSF CRH and CSF NE were significant.
Effects of yohimbine and naloxone on blood pressure, pulse, and behavioral measures
Systolic blood pressure, diastolic blood pressure, mean arterial pressure, and pulse did not increase after the administration of either yohimbine or naloxone. Subjects who received yohimbine had higher anxiety and nervousness scores on the Visual Analog Scales and higher total scores on the PRAS (45) compared with those who received naloxone, but this difference did not reach statistical significance. The peak change in CSF NE after yohimbine administration was positively correlated with the peak change in PRAS score (r = 0.9; P = 0.03) and the peak change in pulse rate (r = 0.97; P = 0.005). However, after correcting for the level of significance required given the number of tests performed, neither of the correlations was significant. None of the other correlations between the biological measures and the cardiovascular or behavioral indexes were significant, even before Bonferroni correction.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The lack of correlation between CSF CRH and plasma cortisol within the group of individuals who received either yohimbine or naloxone supports the view that the concentration of CSF CRH does not reflect HPA axis activity, but is predominantly a measure of non-HPA CRH release. The findings from this study are similar to those of prior studies that suggest that CSF CRH is not derived from the PVN neurons projecting to the median eminence (37, 38, 39, 40, 52). Although the precise source of CSF CRH is unknown, it has been proposed that the CRH neurons in cortical, limbic, and brainstem regions together contribute to the CSF CRH pool because they lie in close neuroanatomical proximity to the ventricular system (37, 39, 40). In contrast, most of the CRH from the PVN of the hypothalamus is believed to enter the circulatory system through the hypothalamo-hypophyseal portal circulation and is reflected in elevated levels of plasma ACTH and cortisol, but not in CSF CRH.
Yohimbine is generally thought to increase the activity of the locus
coeruleus and other noradrenergic projections in the central nervous
system by blocking the NE-mediated tonic inhibition of somatodendritic
2-adrenergic autoreceptors (1, 48). Increased central noradrenergic activity after yohimbine
was reflected in the expected increases in CSF NE concentrations. An
elevation in CSF CRH after yohimbine administration is consistent with
the possibility that central NE has a stimulatory effect on non-HPA CRH
neurons. Significant increases in plasma cortisol after the
administration of yohimbine indicate that noradrenergic inputs exert a
stimulatory effect on PVN CRH neurons that regulate HPA axis activity
(50, 51).
In contrast to yohimbine, naloxone increased plasma cortisol without any suggestion of an increase in CSF CRH. This observation is consistent with recent data supporting a direct action of naloxone on CRH neurons in the PVN of the hypothalamus, with subsequent activation of the pituitary- adrenal axis (32, 33). The lack of an effect of naloxone on CSF CRH suggests that naloxone does not affect non-HPA CRH neurons. This implies, in turn, that opioid receptors are not involved in the regulation of these non-HPA neurons.
When the CSF CRH data are considered together with the measures of plasma cortisol, plasma NE, and CSF NE, it is appears that yohimbine may increase both PVN and non-HPA CRH release in addition to activating central and peripheral noradrenergic systems. The apparent stimulatory effects of yohimbine on non-HPA CRH and central NE are likely to contribute to the anxiogenic properties of yohimbine in humans. In contrast, naloxone, which has similar effects on the HPA axis without affecting CSF NE and CRH concentrations, is not anxiogenic. Although the principal mechanism of yohimbine-induced anxiogenic effects is presumably through its action on NE neurons, it is possible that increases in non-HPA CRH may augment this effect. The small sample size in this study precludes drawing definitive conclusions regarding the role of non-HPA CRH neurons in anxiety in humans. Measurement of CSF CRH and norepinephrine levels 2–3 h after yohimbine administration in a larger group of patients pretreated with either CRH receptor antagonists or placebo may help determine the relative contributions of non-HPA CRH and central NE to yohimbine’s anxiogenic effects. Similar studies in patients with depression and anxiety disorders may shed light on the pathophysiology of CRH dysregulation in these disorders.
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Acknowledgments |
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Footnotes |
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Received June 24, 1999.
Revised January 25, 2000.
Revised July 13, 2000.
Accepted July 19, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2-Adrenergic and opiate receptor blockade.
Synergistic effects on anxiety in healthy subjects. Arch Gen
Psychiatry. 43:1037–1041.[Abstract]
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