Brain Research Bulletin, Vol. 27, pp. 35-39. 0 Pergamon Press plc, 1991. Printed in the U.S.A.
0361-9230/91
$3.00 + II0
Differential Actions of Central Alloxan Upon Opioid and Nonopioid Antinociception in Rats: A Further Examination EDWARD
LUBIN,
BENJAMIN
KEST AND RICHARD
J. BODNAR’
Department of Psychology and Neuropsychology Doctoral Sub-Program, Queens College City University of New York, Flushing, NY 11367 Received
22 January
199 1
LUBIN, E., B. KEST AND R. J. BODNAR. Differential actions of central alloxan upon opioid and nonopioid antinociception in rats: A further examination. BRAIN RBS BULL 27(l) 35-39, 1991 .-Previous work demonstrated that central pretreatment with alloxan significantly reduced antinociception induced by morphine and 2-deoxy-D-glucose (2DG), an opioid-mediated stressor, but not induced by continuous cold-water swims (CCWS), a nonopioid-mediated stressor. The alloxan-induced deficits in 2DG antinociception were ameliorated by coadministration of D-glucose (3 M, 3M-DG). The present study evaluated this relationship further by: a) examining whether central alloxan reduced morphine antinociception following either simultaneous 3M-DG and alloxan coadrninistration, alloxan followed 10 days later by 3M-DG and 3M-DG alone, and b) determining whether central alloxan pretreatment altered nonopioid antinociception induced by the muscarinic cholinergic agonist, pilocarpine. Morphine (2.5-5 mglkg, SC) antinociception on the tail-flick and jump tests was significantly reduced by central alloxan. In contrast, simultaneous coadministration of 3M-DG and alloxan failed to alter morphine antinociception. This ameliorative effect of 3M-DG was not due to its ability to affect morphine antinociception, and was time-dependent in that delays in 3M-DG administration failed to affect the alloxan-induced deficit. Central alloxan pretreatment failed to alter pilocarpine antinociception on the tail-flick test, and increased pilocarpine antinociception on the jump test. That central alloxan reduced opioid (e.g., morphine and 2DG), but not nonopioid (e.g., CCWS, pilocarpine) forms of antinociception suggests a specific mode of action, possibly through disruptions of glucoprivic control mechanisms which is in keeping with the suggestion that opioid systems are sensitive to changes in central glucose function. Antinociception
Alloxan
Morphine
3M D-Glucose
Pilocarpine
Rats
antinociception through a glucosensitive mechanism. This is supported by the ability of 3M D-glucose (3M-DG) to block alloxan-induced diabetes (31,39), and ameliorate the alloxan-induced deficits in 2DG antinociception (21). In contrast, nonopioid antinociception induced by continuous cold-water swims [CCWS: (4,5)] is unaffected by alloxan pretreatment. The present study evaluated further the premise that central alloxan selectively altered opioid and nonopioid antinociception. If alloxan reduces morphine antinociception through a glucosensitive mechanism, then central coadministration of 3M-DG with alloxan should ameliorate such deficits. The fist experiment compared the effects of simultaneous alloxan and 3M-DG coadministration, administration of 3M-DG 10 days following alloxan pretreatment, and 3M-DG itself upon morphine antinociception. The second experiment examined whether central alloxan altered antinociception following the muscarinic receptor agonist, pilocarpine (14,15), with the nonopioid characteristics of the latter confirmed by pretreatment with naloxone.
A role for glucose
in the mediation of morphine antinociception has been hypothesized based on several lines of experimental evidence [see (7)]. Diabetic mice display lower nociceptive thresholds than littermate controls (19), whereas streptozotocintreated mice and rats display reductions in morphine antinociception which is reversed by insulin (33). Fructose and dextrose pretreatment also reduce morphine antinociception (32). Both insulin and naloxone reduce antinociception induced by streptozotocin or glucose-induces hyperglycemia (1). Peripheral alloxan is a pancreatic beta-cell toxin which produces diabetes and hyperglycemia (6, 11, 27). Yet central alloxan at far lower doses fails to produce diabetes or hyperglycemia (29,30), but reduces 2-deoxy-D-glucose (2DG) hyperphagia (23, 29, 38). This latter effect has been attributed to central actions of alloxan upon either brain glucoreceptors (29,38), and/or a glucoprivic control mechanism (28). Central alloxan also significantly reduced the antinociceptive responses following 2DG (21) and morphine (22). Given the partial opioid mediation of 2DG antinociception (3,36), alloxan may be reducing both forms of
‘Requests for reprints should he addressed to Dr. R. J. Bodnar, Department of Psychology, Queens College, CUNY, 65-30 Kissena Blvd., Plushing, NY 11367.
35
36
LUBIN. KEST AND BODNAR
ii t
nal cannula (Plastic Products) which protruded 0.5 mm beyond the tip of the guide cannula. The guide cannula was then removed, the burr hole filled with Gelfoam, and the wound was sutured. All animals were allowed 14 days to recover from surgery, and allow alloxan to exert its effects (23, 29, 30). Following experimental testing, all rats received an overdose of b~it~te (Euthanasia No. 5, H. Schein Co.), were sacrificed, and locaiization of cannula placements was determined microscopically: only animals with correct placements were included in the data analysis.
sol a -.-
:: -r IA. ,
2s -
:’ 4 I-
20
< ;;
15-
g
lo-
a S-
;; c F
0.
3H-06
-c1_
ALLOYAN/3tl-OG
-_cr
ALLOXANM’POG
DELAY
l l
10
I
MORPHINE
o.a-
.E
DOSE (mg/kg)
*
b
0.
5
ALLOYAN
+
4 +
5
;;
CONTROL
+
06-
3
All rats were tested on the tail-flick (8) and jump (IO) tests in mat order. On the tail-flick test, the beam (IITC Company, Woodland Hills, CA) was mounted 8 cm above the dorsum and 3-9 cm proximal to the tip of the rat’s tail with thermal intensity set to produce stable baseline latencies between 2.5 and 4 s. Each session consisted of three latency destinations separated by 10-s intertrlal intervals. To avoid tissue damage, a trial was automatically terminated if a response did not occur within 15 s. On the jump test, electric shock was delivered to the feet of the rat by a shock generator (BRS/LVE) and shock scrambler (Campden Instruments). The jump threshold was defined in mA as the lowest of two consecutive ascending intensities in which the animal simult~eously removed both hindpaws from the grids. Each of the six trials began with the animal receiving a 300-ms footshock at a current intensity of 0.10 mA with subsequent shocks increased in 0.05~mA steps at 10-s intervals until the jump threshold was determined. Protocols
10
I
MORPHINE
DOSE (mg/kg)
FIG. 1. Alterations in morphine antinociception on the tail-flick (upper panel) and jump (lower panel) tests in rats pretreated two weeks earlier with intracerebroventricular (XV) injections of vehicle (control), ailoxan (200 p,g), 3M D-glucose (3M-DG), alloxan and 3M-DG coadministration and alloxan followed 10 days later by 3M-DG (delay). The following is a range of standard errors across the time course for control (0.42-0.80 s; 0.014-0.024 mA), alloxan (0.76-1.16 s; 0.020-0.029 mA). 3M-DG (0.05-0.88 s; 0.015-0.029 mA), alloxan/?lM-DG (O.lO0.94 s; 0.020-0.033 mA) and alloxan/3M-DG delay (0.27-1.43 s; 0.0030.032 mA) conditions. Significant reductions {dark stars) and increases (open stars) in morphine antinociception reiative to vehicle pretreatment are denoted.
METHOD
Subjects, Surgery and Injections Fifty-three male albino Sprague-Dawiey rats (350-500 g) were housed individu~ly and rn~~n~ on a 12-h hght:l2-h dark schedule at ambient temperatures between 22” and 25°C with rat chow and water available ad lib. Following anesthesia with chlorpromazine HCl (3 mg/kg, IP) and ketamine HCl (100 mg/kg, IM), a stainless steel guide cannula (22 gauge, Plastic Products) was stereo&t&ally (Kopf Instruments) positioned so that its tip impinged upon the lateral ventricle at the following coordinates: incisor bar, +5 mm, 0.5 mm anterior to the bregma suture, 1.3 mm lateral to the sagittal suture and 3.6 mm from the top of the skull. An ICV injection (10 ~1 volume) was infused through a Hamilton syringe and polyethylene tubing at a rate of 1 pl every 20 s through a stainless steel 28gauge inter-
Baseline latencies and thresholds were determined over three days prior to and two weeks following injection treatments. In the first protocol, rats received ICV injections of either vehicle (normal saline, n= S), alloxan (200 (*g, Sigma, n= 8), 3M D-glucose (3M-DG, n= 8), alloxan (200 pg) coadministered with 3M-DG (n=8), or alloxan (200 kg) followed by 3M-DC treatment 10 days later (n = 8). Alloxan and D-glucose regimens were chosen for their respective effectiveness to reduce morphine antinociception (22) and to block alloxan-induced deficits in 2DG antinociception (21) and hyperphagia (23,29). Two weeks after treatment, each group received the following three injection conditions: vehicle (1 ml normal &n&g body weight, SC) and morphine (Pennick Laboratories) at doses of 2.5 and 5.0 mgkg. Tail-flick latencies and jump thresholds were assessed 30 and 60 min thereafter. This regimen yielded significant alloxaninduced deficits in morphine antinociception previously (22). In the second protocol, rats received ICV injections of either vehicle (normal saline, n=8) or alloxan (200 pg, n= 8). Two weeks after treatment, each group received five injection conditions: vehicle (1 ml normal saline/kg body weight, IP), pilocarpine (Sigma) at doses of 0.5, 2.0 and 5.0 mg/kg, and naloxone (Sigma, mg/kg, IP) 15 min prior to pilocarpine (5 mg/ kg, IP). Latencies and thresholds were assessed 30, 60, 90 and 120 min thereafter. Statistical Analyses Split-plot analyses of variance assessed sig~~c~t effects among pretreatments and drug conditions with DuMett comparisons used to discern differences between vehicle and either morphine or pilocarpine. Dunn comparisons evaluated differences among pretreatment conditions. To evaluate pretreatment effects
37
CENTRAL ALLOXAN AND ANTINOCICEI’TION
;i
5
+
ColrrnoL *LLox*N
l
?
/t -_t
-b ‘.OO
2 5
0.50-
ii i *
0.25-
a
E I- 0.750.00 ~ k
..J.._
.
..J..._
---I
IO PILOCARPINE
1
I
PILOCARPINE
DOSE (mg/kg)
10
DOSE (mglkg)
FIG. 2. Alterations across the pilocarpine antinociception on the dose-response curve on the tail-flick (left panel) and jump (right panel) tests following ICV pretreatment with alloxan. Significant potentiations in pilocarpine antinociception following alloxan relative to control pretreatment are denoted (dark star). The following is a range of standard errors across the dose-response curve for vehicle (0.07-1.36 s; 0.009~.046 mA) and alloxan (0.07-1.35 s; 0.008~.080 mA) treatments.
cantly reduced morphine antinociception on the tail-flick (Fig. la, 2.5 mgikg: 45%; 5 mg/kg: 35%) and jump (Fig. lb, 2.5 mg/kg: 48%; 5 mg/kg: 34%) tests. Simultaneous coadministration of alloxan with 3M-DG completely abolished the alloxaninduced deficits in morphine antinociception on the tail-flick test, and significantly increased morphine antinociception on the jump test following the 2.5 (25%) and 5 (11%) mg/kg doses. The ameliorating actions of 3M-DG were not due to its actions upon morphine antinociception. Further, if 3M-DG was administered 10 days after alloxan treatment, alloxan again significantly reduced morphine antinociception on the tail-flick (2.5 mg/kg: 57%; 5 mg/kg: 40%) and jump (2.5 mglkg: 535; 5 mg/ kg: 40%) tests.
upon morphine and pilocarpine antinociception, all figures are expressed in terms of total analgesic difference scores which were derived by subtracting each postdrug effect at each time point from its corresponding vehicle value, and summing the difference. RESULTS
Protocol 1 -Alloxan,
3M-DG and Morphine Antinociception
Significant differences in latencies and thresholds were observed among central treatments (tail-flick: p
0361-9230/91
$3.00 + II0
Differential Actions of Central Alloxan Upon Opioid and Nonopioid Antinociception in Rats: A Further Examination EDWARD
LUBIN,
BENJAMIN
KEST AND RICHARD
J. BODNAR’
Department of Psychology and Neuropsychology Doctoral Sub-Program, Queens College City University of New York, Flushing, NY 11367 Received
22 January
199 1
LUBIN, E., B. KEST AND R. J. BODNAR. Differential actions of central alloxan upon opioid and nonopioid antinociception in rats: A further examination. BRAIN RBS BULL 27(l) 35-39, 1991 .-Previous work demonstrated that central pretreatment with alloxan significantly reduced antinociception induced by morphine and 2-deoxy-D-glucose (2DG), an opioid-mediated stressor, but not induced by continuous cold-water swims (CCWS), a nonopioid-mediated stressor. The alloxan-induced deficits in 2DG antinociception were ameliorated by coadministration of D-glucose (3 M, 3M-DG). The present study evaluated this relationship further by: a) examining whether central alloxan reduced morphine antinociception following either simultaneous 3M-DG and alloxan coadrninistration, alloxan followed 10 days later by 3M-DG and 3M-DG alone, and b) determining whether central alloxan pretreatment altered nonopioid antinociception induced by the muscarinic cholinergic agonist, pilocarpine. Morphine (2.5-5 mglkg, SC) antinociception on the tail-flick and jump tests was significantly reduced by central alloxan. In contrast, simultaneous coadministration of 3M-DG and alloxan failed to alter morphine antinociception. This ameliorative effect of 3M-DG was not due to its ability to affect morphine antinociception, and was time-dependent in that delays in 3M-DG administration failed to affect the alloxan-induced deficit. Central alloxan pretreatment failed to alter pilocarpine antinociception on the tail-flick test, and increased pilocarpine antinociception on the jump test. That central alloxan reduced opioid (e.g., morphine and 2DG), but not nonopioid (e.g., CCWS, pilocarpine) forms of antinociception suggests a specific mode of action, possibly through disruptions of glucoprivic control mechanisms which is in keeping with the suggestion that opioid systems are sensitive to changes in central glucose function. Antinociception
Alloxan
Morphine
3M D-Glucose
Pilocarpine
Rats
antinociception through a glucosensitive mechanism. This is supported by the ability of 3M D-glucose (3M-DG) to block alloxan-induced diabetes (31,39), and ameliorate the alloxan-induced deficits in 2DG antinociception (21). In contrast, nonopioid antinociception induced by continuous cold-water swims [CCWS: (4,5)] is unaffected by alloxan pretreatment. The present study evaluated further the premise that central alloxan selectively altered opioid and nonopioid antinociception. If alloxan reduces morphine antinociception through a glucosensitive mechanism, then central coadministration of 3M-DG with alloxan should ameliorate such deficits. The fist experiment compared the effects of simultaneous alloxan and 3M-DG coadministration, administration of 3M-DG 10 days following alloxan pretreatment, and 3M-DG itself upon morphine antinociception. The second experiment examined whether central alloxan altered antinociception following the muscarinic receptor agonist, pilocarpine (14,15), with the nonopioid characteristics of the latter confirmed by pretreatment with naloxone.
A role for glucose
in the mediation of morphine antinociception has been hypothesized based on several lines of experimental evidence [see (7)]. Diabetic mice display lower nociceptive thresholds than littermate controls (19), whereas streptozotocintreated mice and rats display reductions in morphine antinociception which is reversed by insulin (33). Fructose and dextrose pretreatment also reduce morphine antinociception (32). Both insulin and naloxone reduce antinociception induced by streptozotocin or glucose-induces hyperglycemia (1). Peripheral alloxan is a pancreatic beta-cell toxin which produces diabetes and hyperglycemia (6, 11, 27). Yet central alloxan at far lower doses fails to produce diabetes or hyperglycemia (29,30), but reduces 2-deoxy-D-glucose (2DG) hyperphagia (23, 29, 38). This latter effect has been attributed to central actions of alloxan upon either brain glucoreceptors (29,38), and/or a glucoprivic control mechanism (28). Central alloxan also significantly reduced the antinociceptive responses following 2DG (21) and morphine (22). Given the partial opioid mediation of 2DG antinociception (3,36), alloxan may be reducing both forms of
‘Requests for reprints should he addressed to Dr. R. J. Bodnar, Department of Psychology, Queens College, CUNY, 65-30 Kissena Blvd., Plushing, NY 11367.
35
36
LUBIN. KEST AND BODNAR
ii t
nal cannula (Plastic Products) which protruded 0.5 mm beyond the tip of the guide cannula. The guide cannula was then removed, the burr hole filled with Gelfoam, and the wound was sutured. All animals were allowed 14 days to recover from surgery, and allow alloxan to exert its effects (23, 29, 30). Following experimental testing, all rats received an overdose of b~it~te (Euthanasia No. 5, H. Schein Co.), were sacrificed, and locaiization of cannula placements was determined microscopically: only animals with correct placements were included in the data analysis.
sol a -.-
:: -r IA. ,
2s -
:’ 4 I-
20
< ;;
15-
g
lo-
a S-
;; c F
0.
3H-06
-c1_
ALLOYAN/3tl-OG
-_cr
ALLOXANM’POG
DELAY
l l
10
I
MORPHINE
o.a-
.E
DOSE (mg/kg)
*
b
0.
5
ALLOYAN
+
4 +
5
;;
CONTROL
+
06-
3
All rats were tested on the tail-flick (8) and jump (IO) tests in mat order. On the tail-flick test, the beam (IITC Company, Woodland Hills, CA) was mounted 8 cm above the dorsum and 3-9 cm proximal to the tip of the rat’s tail with thermal intensity set to produce stable baseline latencies between 2.5 and 4 s. Each session consisted of three latency destinations separated by 10-s intertrlal intervals. To avoid tissue damage, a trial was automatically terminated if a response did not occur within 15 s. On the jump test, electric shock was delivered to the feet of the rat by a shock generator (BRS/LVE) and shock scrambler (Campden Instruments). The jump threshold was defined in mA as the lowest of two consecutive ascending intensities in which the animal simult~eously removed both hindpaws from the grids. Each of the six trials began with the animal receiving a 300-ms footshock at a current intensity of 0.10 mA with subsequent shocks increased in 0.05~mA steps at 10-s intervals until the jump threshold was determined. Protocols
10
I
MORPHINE
DOSE (mg/kg)
FIG. 1. Alterations in morphine antinociception on the tail-flick (upper panel) and jump (lower panel) tests in rats pretreated two weeks earlier with intracerebroventricular (XV) injections of vehicle (control), ailoxan (200 p,g), 3M D-glucose (3M-DG), alloxan and 3M-DG coadministration and alloxan followed 10 days later by 3M-DG (delay). The following is a range of standard errors across the time course for control (0.42-0.80 s; 0.014-0.024 mA), alloxan (0.76-1.16 s; 0.020-0.029 mA). 3M-DG (0.05-0.88 s; 0.015-0.029 mA), alloxan/?lM-DG (O.lO0.94 s; 0.020-0.033 mA) and alloxan/3M-DG delay (0.27-1.43 s; 0.0030.032 mA) conditions. Significant reductions {dark stars) and increases (open stars) in morphine antinociception reiative to vehicle pretreatment are denoted.
METHOD
Subjects, Surgery and Injections Fifty-three male albino Sprague-Dawiey rats (350-500 g) were housed individu~ly and rn~~n~ on a 12-h hght:l2-h dark schedule at ambient temperatures between 22” and 25°C with rat chow and water available ad lib. Following anesthesia with chlorpromazine HCl (3 mg/kg, IP) and ketamine HCl (100 mg/kg, IM), a stainless steel guide cannula (22 gauge, Plastic Products) was stereo&t&ally (Kopf Instruments) positioned so that its tip impinged upon the lateral ventricle at the following coordinates: incisor bar, +5 mm, 0.5 mm anterior to the bregma suture, 1.3 mm lateral to the sagittal suture and 3.6 mm from the top of the skull. An ICV injection (10 ~1 volume) was infused through a Hamilton syringe and polyethylene tubing at a rate of 1 pl every 20 s through a stainless steel 28gauge inter-
Baseline latencies and thresholds were determined over three days prior to and two weeks following injection treatments. In the first protocol, rats received ICV injections of either vehicle (normal saline, n= S), alloxan (200 (*g, Sigma, n= 8), 3M D-glucose (3M-DG, n= 8), alloxan (200 pg) coadministered with 3M-DG (n=8), or alloxan (200 kg) followed by 3M-DC treatment 10 days later (n = 8). Alloxan and D-glucose regimens were chosen for their respective effectiveness to reduce morphine antinociception (22) and to block alloxan-induced deficits in 2DG antinociception (21) and hyperphagia (23,29). Two weeks after treatment, each group received the following three injection conditions: vehicle (1 ml normal &n&g body weight, SC) and morphine (Pennick Laboratories) at doses of 2.5 and 5.0 mgkg. Tail-flick latencies and jump thresholds were assessed 30 and 60 min thereafter. This regimen yielded significant alloxaninduced deficits in morphine antinociception previously (22). In the second protocol, rats received ICV injections of either vehicle (normal saline, n=8) or alloxan (200 pg, n= 8). Two weeks after treatment, each group received five injection conditions: vehicle (1 ml normal saline/kg body weight, IP), pilocarpine (Sigma) at doses of 0.5, 2.0 and 5.0 mg/kg, and naloxone (Sigma, mg/kg, IP) 15 min prior to pilocarpine (5 mg/ kg, IP). Latencies and thresholds were assessed 30, 60, 90 and 120 min thereafter. Statistical Analyses Split-plot analyses of variance assessed sig~~c~t effects among pretreatments and drug conditions with DuMett comparisons used to discern differences between vehicle and either morphine or pilocarpine. Dunn comparisons evaluated differences among pretreatment conditions. To evaluate pretreatment effects
37
CENTRAL ALLOXAN AND ANTINOCICEI’TION
;i
5
+
ColrrnoL *LLox*N
l
?
/t -_t
-b ‘.OO
2 5
0.50-
ii i *
0.25-
a
E I- 0.750.00 ~ k
..J.._
.
..J..._
---I
IO PILOCARPINE
1
I
PILOCARPINE
DOSE (mg/kg)
10
DOSE (mglkg)
FIG. 2. Alterations across the pilocarpine antinociception on the dose-response curve on the tail-flick (left panel) and jump (right panel) tests following ICV pretreatment with alloxan. Significant potentiations in pilocarpine antinociception following alloxan relative to control pretreatment are denoted (dark star). The following is a range of standard errors across the dose-response curve for vehicle (0.07-1.36 s; 0.009~.046 mA) and alloxan (0.07-1.35 s; 0.008~.080 mA) treatments.
cantly reduced morphine antinociception on the tail-flick (Fig. la, 2.5 mgikg: 45%; 5 mg/kg: 35%) and jump (Fig. lb, 2.5 mg/kg: 48%; 5 mg/kg: 34%) tests. Simultaneous coadministration of alloxan with 3M-DG completely abolished the alloxaninduced deficits in morphine antinociception on the tail-flick test, and significantly increased morphine antinociception on the jump test following the 2.5 (25%) and 5 (11%) mg/kg doses. The ameliorating actions of 3M-DG were not due to its actions upon morphine antinociception. Further, if 3M-DG was administered 10 days after alloxan treatment, alloxan again significantly reduced morphine antinociception on the tail-flick (2.5 mg/kg: 57%; 5 mg/kg: 40%) and jump (2.5 mglkg: 535; 5 mg/ kg: 40%) tests.
upon morphine and pilocarpine antinociception, all figures are expressed in terms of total analgesic difference scores which were derived by subtracting each postdrug effect at each time point from its corresponding vehicle value, and summing the difference. RESULTS
Protocol 1 -Alloxan,
3M-DG and Morphine Antinociception
Significant differences in latencies and thresholds were observed among central treatments (tail-flick: p
