Comparison of the effects of clonidine and yohimbine on pupillary diameter at different illumination levels

Abstract

Aims

To evaluate the pupillary effects of single doses of the α₂-adrenoceptor agonist clonidine and the α₂-adrenoceptor antagonist yohimbine under several illumination conditions.

Methods

Sixteen healthy male volunteers received clonidine 0.2 mg, yohimbine 22 mg, clonidine 0.2 mg + yohimbine 22 mg in a double-blind placebo-controlled, cross-over study. 2 h post drug ingestion pupil diameter was recorded in darkness, and at luminance levels of 6 Cd m⁻², 91 Cd m⁻² and 360 Cd m⁻². The effects of the active treatments on pupil diameter were also expressed as the differences from the placebo condition (‘placebo-corrected’ data; mean [95% CI]).

Results

Clonidine had little effect on pupil diameter in darkness; however, it caused a significant, light-dependent, miosis when the eye was illuminated. On the other hand yohimbine increased pupil size; this increase was significant at 91 and 360 Cd m⁻². There were no significant differences between the effects of the combined treatment (clonidine 0.2 mg + yohimbine 22 mg) and the effect of placebo.

Conclusions

The pupillary effects of clonidine and yohimbine are likely to reflect the interaction of these drugs with inhibitory α₂-adrenoceptors located on central noradrenergic neurones, which in turn would lead to a decrease and an increase, respectively, in sympathetic outflow to the iris. The light dependence of the pupillary effects of these drugs, however, suggests that the parasympathetic light reflex pathway is also involved, which is known to be under inhibitory control from the central noradrenergic neurones. Modulation of parasympathetic outflow seems to play an important role since both drugs had relatively little effect on pupil diameter in darkness when sympathetic activity predominates.

Introduction

In humans the systemic administration of the α₂-adrenoceptor agonist clonidine evokes a miotic response [1–5], whilst administration of the α₂-adrenoceptor antagonist yohimbine evokes mydriasis [6]. Furthermore, the clonidine-evoked miosis is susceptible to antagonism by α₂-adrenoceptor antagonists [1, 3]. In a recent study Bitsios et al. [4] observed that the miosis evoked by clonidine was greater in light than in darkness. In the present experiment we extended this observation by comparing the effects of clonidine on pupil size both in darkness and at three different levels of illumination. Furthermore, we examined the effects of yohimbine, and the combination of clonidine and yohimbine, under the same conditions.

Methods

Subjects

Sixteen healthy drug-free male volunteers aged 21.6 ± 0.4 years (mean ± s.e. mean) and weighing 82.2 ± 2.3 kg (mean ± s.e. mean) participated. Each subject completed a brief medical history and underwent a complete physical examination before inclusion in the study. All subjects reported compliance with the request to abstain from caffeine-containing beverages on each test day. All volunteers gave their written informed consent following a verbal explanation of the study and after reading a detailed information sheet. The study protocol was approved by the University of Nottingham Medical School Ethics Committee.

Drugs

Clonidine hydrochloride (0.2 mg), yohimbine hydrochloride (22 mg), clonidine hydrochloride (0.2 mg) + yohimbine hydrochloride (22 mg), and placebo were prepared in identical capsules and administered orally.

Design

Each subject participated in four experimental sessions at weekly intervals. Subjects were allocated to treatments and sessions according to a double-blind, balanced, cross-over design.

Pupillometry and procedure

The timing of the measurement of pupil diameter after the ingestion of the capsule was based on reports in the literature regarding the time required for the development of peak effects following single oral doses of clonidine and yohimbine. The maximum plasma level after a single dose of clonidine has been reported to require 60–90 min to develop [7], however, the maximum pharmacodynamic effects take 2–4 h to develop [8]. Although the peak plasma concentration of yohimbine after a single oral dose appears under 1 h [9], maximum pharmacodynamic effects take approximately 2 h to develop [10]. Two hours after the ingestion of the capsule, subjects were placed in a darkened room and positioned in front of a portable binocular infrared video pupillometer (Procyon, UK). A calibrated internal light source within the pupillometer allowed pupil diameter to be recorded under four illumination conditions: darkness; 6 Cd m⁻²; 91 Cd m⁻² and 360 Cd m⁻². Pupil diameter was first recorded in darkness and then recorded under each of the increasing light intensities for 2 s at 4 Hz and stored to disk for off-line analysis. All measurements were obtained between 12.00 and 17.00 h.

Data analysis

Mean pupil size under each illumination condition was subjected to two-way analysis of variance (treatment × illumination condition) with repeated measures on both factors. Individual comparisons of active treatments and placebo (at each illumination condition) were made using Dunnett's t-test with an a priori criterion of P < 0.05 (d.f. = 135; k = 4; criterion t = 2.38). In addition, each individual's response under the placebo condition was subtracted from the response obtained under each of the active treatments in order to reveal the size of the response evoked by the active drug (‘placebo-corrected response’). The effects of the active treatments were analysed by comparing placebo-corrected responses with a zero baseline (i.e. placebo-corrected response for placebo) with multiple t-tests using Bonferroni's correction with an a priori criterion of P < 0.05 (d.f. = 15; criterion t = 3.29).

Results

Absolute pupil diameter

Table 1 shows pupil diameter after each drug treatment condition under the four illumination conditions. Two-way anova revealed significant main effects of treatment (F_3,45 = 18.26, P < 0.0001) and illumination condition (F_3,45 = 294.6, P < 0.0001) and a significant treatment × illumination condition interaction (F_9,135 = 4.73, P < 0.0001). Individual comparisons with placebo revealed that yohimbine evoked a significant mydriasis at each illumination level (darkness t = 3.1, 6 Cd m⁻² t = 2.9, 91 Cd m⁻² t = 4.0 and 360 Cd m⁻² t = 3.8), whilst clonidine evoked miosis that was significant at 6 Cd m⁻² (t = 5.06), 91 Cd m⁻² (t = 7.35) and 360 Cd m⁻² (t = 3.43), but not in darkness (t = 0.3). Pupil size after the combined drug treatment (clonidine + yohimbine) did not differ significantly from that recorded after placebo at any of the illumination levels tested (t < 2.0 at all illumination levels).

Table 1.

Pupil diameter following the administration of the four treatments.

Placebo-corrected data

Figure 1 Table 1 show the placebo-corrected pupil diameter data following the administration of active drug treatments. Significant differences from baseline (‘placebo’) level were obtained after treatment with clonidine under ambient illumination of 6 Cd m⁻² (t = 3.53), 91 Cd m⁻² (t = 6.05) and 360 Cd m⁻² (t = 3.88). Placebo-corrected pupil size after yohimbine was significantly different from baseline at the 91 Cd m⁻² (t = 3.35) and 360 Cd m⁻² (t = 3.56). Placebo-corrected pupil size after the combined drug treatment (clonidine + yohimbine), did not differ significantly from baseline at any of the illumination levels tested in this experiment (in all cases t < 2.1).

Figure 1.

Relationship between level of luminance and placebo-corrected pupil diameter (mm). Each point corresponds to the mean (n = 16) at four luminance levels (darkness, 6 Cd m⁻², 91 Cd m⁻² and 360 Cd m⁻²), plotted on a logarithmic scale. Horizontal broken line corresponds to the placebo baseline (‘placebo-corrected’ placebo values). Open circles: placebo; open triangles: clonidine 0.2 mg; closed triangles: yohimbine 22 mg; closed circles: clonidine 0.2 mg + yohimbine 22 mg. Statistically significant differences from placebo *P < 0.05, **P < 0.01.

Discussion

In the present experiment clonidine, an α₂-adrenoceptor agonist, had no effect on pupil diameter in the dark, but had a clear miotic effect in the light. Furthermore the size of the miosis appeared to be dependent on the level of illumination, higher illumination levels being associated with larger miotic responses. It should be noted that this relationship did not hold for the highest level of illumination, probably reflecting the fact that the level to which the pupil could be constricted was approaching the smallest possible pupil diameter (‘floor effect’; see [11, 12]). Although a quantitative relationship between the level of illumination and the size of the miotic response to clonidine has not been demonstrated previously, the present results are in good qualitative agreement with previous reports in the literature. Firstly, the effect of clonidine on the pupil in the dark has been reported to be either nonobservable [6], or very small [4], and most reports of a miotic effect of clonidine are based on experiments conducted in an illuminated room [1–3]. Secondly, it has been reported that the size of the miotic response to clonidine in the same group of subjects is larger when the measurement is carried out under illumination rather than in the dark [4].

As the α₂-adrenoceptor antagonist yohimbine usually has effects on autonomic functions which are opposite to those evoked by clonidine [13], we also examined the effect of yohimbine on the pupil. In contrast to clonidine, yohimbine tended to increase the size of the pupil. This effect of yohimbine was present in the dark, but became somewhat accentuated at the higher illumination levels. The mydriatic effect of yohimbine in the dark is in agreement with the previous report of Morley et al. [6].

The most likely explanation for the pupillary effects of clonidine and yohimbine is that they are related to the interaction of these drugs with α₂-adrenoceptors. Indeed, in the present study, when the two drugs were applied together they tended to cancel out each others' effects. Although these drugs interact with α₂-adrenoceptors both in the periphery and the central nervous system, it is generally accepted that their overall effect in healthy humans mainly reflects the stimulation and antagonism, respectively,of α₂-adrenoceptors located on central noradrenergic neurones in the brainstem. The stimulation of inhibitory α₂-adrenoceptors by clonidine would reduce the activity of these neurones leading to a sympatholytic effect, whereas yohimbine would increase the activity of noradrenergic neurones via the blockade of the same somatodendritic autoreceptors resulting in sympathetic activation [13].

The most plausible explanation for the miotic effect of clonidine and the mydriatic effect of yohimbine is that they result from sympathetic inhibition and excitation by these drugs, respectively. This mechanism, however, cannot account for the light dependence of the pupillary effect of these drugs since the effect of light on the pupil is mediated mainly by the parasympathetic input to the iris [14]. It is known, however, that the central noradrenergic neurones innervate the Edinger-Westphal nucleus, the preganglionic parasympathetic nucleus controlling pupil constriction, and it is likely that this innervation results in a tonic inhibition of the light reflex by the noradrenergic neurones [13]. Therefore, the stimulation of inhibitory α₂-adrenoceptors on central noradrenergic neurones by clonidine is expected to result in a dual effect: reduction in sympathetic outflow and increase in parasympathetic outflow to the iris, of which only the second effect would depend on the level of illumination. Conversely, by antagonizing α₂-adrenoceptors on central noradrenergic neurones, yohimbine would increase sympathetic activity and reduce parasympathetic outflow, both effects leading to mydriasis.

In conclusion, the present results show that the pupillary effects of clonidine and yohimbine are likely to be mediated by both the sympathetic and parasympathetic inputs to the iris. Furthermore, the modulation of the parasympathetic outflow seems to play the dominant role since both clonidine and yohimbine had relatively small effects on pupil diameter in the dark when pupil diameter is almost entirely determined by sympathetic activity [15]. These results have methodological relevance for studies in which the pupillary effects of clonidine and yohimbine are used in models of human anxiety (e.g. ‘fear-inhibited light reflex’ [5]).