www.elsevier.com/locate/brainres

Brain Research 1014 (2004) 244–246

Short communication

Sex differences in systemic morphine analgesic tolerance following

intrathecal morphine injections

Eileen Hopkinsa, Grace Rossib, Benjamin Kesta,c,*

aNeuropsychology Doctoral Subprogram, Queens College, City University of New York, Flushing, NY 11367, USAbDepartment of Psychology, C.W. Post College, Long Island University, Brookville, NY 11548, USA

cDepartment of Psychology and Center for Developmental Neuroscience, College of Staten Island, City University of New York,

Staten Island, NY 10314, USA

Accepted 31 March 2004

Available online 28 May 2004

Abstract

Morphine analgesic potency following systemic administration was assessed in male and female mice undergoing prior and repeated

intrathecal morphine injections. Although morphine ED50 values were significantly increased in both sexes relative to their respective saline-

injected controls, the magnitude of tolerance was greater in females. Intrathecal injection alone had no effect on morphine analgesia. The data

suggest that spinal mechanisms contribute to sex differences in analgesic tolerance following systemic morphine administration.

D 2004 Elsevier B.V. All rights reserved.

Theme: Neurotransmitters, modulators, transporters, and receptors

Topic: Opioids: anatomy, physiology, and behavior

Keywords: Morphine; Tolerance; Sex difference; Spinal cord

The difference between males and females in analgesic

effect following morphine administration has deservedly

received notable attention [9]. Although the mechanism of

this sexual diergism remains unclear, the persistence of

analgesic sex differences after central morphine microin-

jection in mice [7] and rats [5] clearly implicates contri-

butions from sites within the CNS. Like morphine

analgesia, morphine analgesic tolerance is also subject to

sex differences in rats and mice [1,3,8 (see however Ref.

[2])]. In the mouse, we have previously reported that the

reduced analgesic potency of morphine is of greater

magnitude in female mice following 4 or 8 days of

repeated systemic injection [8]. Since morphine analgesia

can be elicited after either spinal [14] or supraspinal [5–7]

application, both sites can contribute to analgesia following

systemic morphine injection. Thus, although the mecha-

nism contributing to morphine tolerance has yet to be

0006-8993/$ - see front matter D 2004 Elsevier B.V. All rights reserved.

doi:10.1016/j.brainres.2004.03.056

* Corresponding author. Department of Psychology, and Center for

Developmental Neuroscience, City University of New York, College of

Staten Island, 2800 Victory Blvd., Staten Island, NY 10314, USA. Tel.: +1-

718-982-4070; fax: +1-718-982-4114.

E-mail address: kest@postbox.csi.cuny.edu (B. Kest).

clearly elucidated, tolerance to the analgesic effect of

systemic morphine injection may result from alterations

at either, or both, CNS loci. Similarly, sex differences in

analgesic tolerance to morphine following its systemic

injection in mice may also reflect the differential develop-

ment of tolerance between males and females in morphine

analgesic mechanisms located spinally, supraspinally, or

both. We have previously studied the contribution of

supraspinal mechanisms to sex differences in tolerance

by comparing the morphine analgesia dose– response

curves of male and female mice following chronic intra-

cerebroventricular morphine injection. Although increased

ED50 values were observed for both sexes—demonstrative

of analgesic tolerance—there was no significant difference

between males and females [6].

The aim of the present study was to now assess the

contribution of spinal morphine analgesic mechanisms to

sex differences in systemic morphine analgesic tolerance.

Using the same CD-1 strain of mouse from previous studies,

cumulative dose–response curves for systemic morphine

analgesia were generated and derived ED50 values com-

pared for males and females after repeated intrathecal

morphine microinjection.

Table 1

Analgesic potency of systemic morphine injection in male and female mice

following repeated intrathecal morphine injections

Sex (n) Treatment Test

day

Baseline

latencyaED50

b 95% CIc

Male (10) Naı̈ve 1 2.7 (F 0.7) 3.0 1.7–4.9

Female (10) Naı̈ve 1 2.6 (F 0.6) 4.2 2.5–6.6

Male (12) Saline 4 2.5 (F 0.7) 2.0 1.3–3.0

Female (12) Saline 4 2.3 (F 0.3) 3.1 2.0–4.6

Male (16) Morphine 4 1.9 (F 0.5)d 10.3e 7.2–14.3

Female (16) Morphine 4 2.0 (F 0.7)d 21.9e 15.7–30.4

a Latency on tail withdrawal test (sec.F S.E.M) prior to dose– response

study.b Values are morphine dose (mg/kg) to produce analgesia in 50% of

subjects.c 95% confidence intervals.d Denotes significant reduction in latencies relative to same-sex saline-

treated and naı̈ve controls.e Denotes significant reduction in analgesic potency relative to same-

sex saline-treated and naı̈ve controls.

E. Hopkins et al. / Brain Research 1014 (2004) 244–246 245

Adequate measures were taken to minimize subject pain

or discomfort throughout. Thus, under oxygen/isoflourane

inhalant anesthesia, adult male and female mice were

injected intrathecally with a 2 Al bolus (lower lumbar to

upper-sacral region) of morphine sulfate (National Institute

on Drug Abuse, Rockville, MD) three times a day for 3 days

using an escalating dosing schedule (10, 20, and 40 Ag on

Days 1, 2, and 3, respectively). Control mice received

equivalent injections of vehicle (0.9% physiological saline).

For 10–20% of mice of both sexes, intrathecal injection of

the highest morphine dose produced lethality or hyperflex-

ion/seizure-like activity in hind limbs for a period of f 20

min. These animals were excluded from the study. No such

effects were observed in vehicle-treated mice.

On Day 4, baseline nociceptive sensitivity was assessed

on the tail-withdrawal test, chosen because of its stability

after repeated measurement [6–8]. Each animal’s latency to

a reflexive withdrawal of the tail from water maintained at a

noxious temperature (49 jC) was measured twice to the

nearest 0.1 s and averaged. A cut-off latency of 15 s was

employed to prevent possible tissue damage. Morphine

analgesic potency in male and female mice was compared

using ED50 values derived from cumulative dose–response

curves. Specifically, all mice were subcutaneously injected

with a 2 mg/kg dose of morphine and withdrawal latencies

reassessed 30 min later. Animals not analgesic were injected

with the next in a series of increasing (f 0.25 log) mor-

phine doses (3.6, 6.5, 11.7, and 21 mg/kg) and retested 30

min later. Analgesia was operationally defined as a doubling

of each subject’s mean baseline withdrawal latency on

consecutive determinations at a given dose. In addition to

morphine-treated and vehicle-treated control mice, cumula-

tive dose–response curves were also generated for a group

of naı̈ve mice receiving no prior intrathecal injections.

Two-way (sex� treatment) ANOVA was also used to

compare baseline tail-withdrawal latencies (a= 0.05). Mor-

phine dose–response data were analyzed using the BLISS-

21 computer program, which provides ED50 values and 95%

confidence intervals (CI) [11]. Non-overlapping 95% CIs

are considered significantly different.

Table 1 presents baseline withdrawal latencies and ED50

values for morphine analgesia after systemic injection in

male and female mice receiving repeated intrathecal injec-

tions for 3 days prior to testing. Repeated intrathecal

morphine injections significantly reduced baseline with-

drawal latencies on Day 4 relative to saline-injected and

Day 1 naı̈ve control groups, a finding consistent with the

literature on opioid-induced hyperalgesia [12]. There was

however no significant interaction of sex within this or any

other treatment group. Potency estimates for males and

females receiving intrathecal vehicle injections did not

differ, indicating no initial sex difference in systemic mor-

phine analgesic potency. Furthermore, the ED50 values for

this group did not significantly differ from those obtained in

same sex naı̈ve mice, demonstrating that the repeated

handling/stress associated with intrathecal injections did

not confound analgesic assessment. In contrast to their

respective vehicle-injected controls, repeated intrathecal

morphine injections significantly increased ED50 values in

male and female mice, indicating analgesic tolerance. How-

ever, the magnitude of tolerance was not equal between

sexes. Although ED50 values were increased five-fold in

males relative to saline-injected controls, non-overlapping

95% CIs between male and female mice receiving intrathe-

cal morphine injections indicate that the greater seven-fold

increase in female ED50 values relative to saline-injected

controls was indeed significantly larger, demonstrating a

potency loss of greater magnitude in this sex.

The present findings are highly consistent with sex

differences observed for analgesic tolerance after 3 days

of systemic morphine administration [8] with regards to

both direction (tolerance: females > males) and approxi-

mate magnitude (f two-fold). In contrast, repeated intra-

cerebroventricular morphine injections produced significant

analgesic tolerance of equal magnitude in male and female

mice [6]. Collectively, these studies strongly suggest a

spinal locus for sex differences in analgesic tolerance to

morphine following systemic administration in the mouse.

Future studies should address the relevance of this CNS

locus to sex differences in morphine tolerance in rats, where

sex differences are not uniformly observed and have been

studied after systemic morphine administration only [1–3].

A possible mechanism whereby morphine repeatedly

applied to the spinal cord affects systemic analgesic toler-

ance sex-dependently is suggested with great reservation for

two reasons. First, the mechanism underlying morphine

analgesic tolerance for any sex and at any level of the

neuraxis is not well understood [4]. Second, although

gonadal status and hormonal levels can influence morphine

analgesia in the spinal cord, these findings do not yet

provide a consistent and cogent explanation of sex differ-

ences in analgesia [9], and in any case there are no data

relating these variables to morphine analgesic tolerance.

E. Hopkins et al. / Brain Research 1014 (2004) 244–246246

Consequently, speculation about the role of sex hormones is

clearly beyond the scope of the present results. We are

intrigued, however, by the demonstration in mice [10,13]

and rats [15] that sub-analgesic doses of morphine co-

administered spinally and supraspinally act synergistically

in the production of analgesia. In addition, it has been

shown that the synergism between spinal and supraspinal

sites are reduced to additive effects when mice are rendered

tolerant to morphine following repeated systemic adminis-

tration [10]. If the synergistic relationship between spinal

and supraspinal sites is subject to sex differences, this would

not only account for sex differences in morphine analgesia,

but would be logically consistent with both the present

results and our previous observations on sex differences in

morphine analgesic tolerance after systemic administration

[8]. This possibility should be directly assessed in future

studies by directly comparing spinal and supraspinal mor-

phine analgesic synergy in male and female mice.

Acknowledgements

This work was supported by PSC/CUNY and CSI/IBR

Center for Developmental Neuroscience.

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