A lateralized deficit in morphine antinociception after unilateral inactivation of the central amygdala

Abstract

The amygdala is a forebrain region that is receiving increasing attention as a modulator of pain sensation. The amygdala contributes to antinociception elicited by both psychological factors (e.g., fear) and exogenous opioid agonists. Unlike the midbrain periaqueductal gray matter (PAG) or rostral ventromedial medulla, the amygdala is a pain-modulating region that has clear bilateral representation in the brain, making it possible to determine whether pain-modulating effects of this region are lateralized with respect to the peripheral origin of noxious stimulation. Unilateral inactivation of the central nucleus of the amygdala (Ce) plus adjacent portions of the basolateral amygdaloid complex (with either the excitotoxin NMDA or the GABAA agonist muscimol) reduced the ability of morphine to suppress prolonged, formalin-induced pain derived from the hindpaw ipsilateral, but not contralateral, to the inactivated region. This effect was evident regardless of the nociceptive scoring method used (weighted scores or flinch-frequency method) and was not accompanied by a concurrent reduction in morphine-induced hyperlocomotion. Unilateral lesions restricted to the basolateral amygdaloid complex (i.e., not including the Ce) did not reduce the ability of morphine to suppress formalin-induced pain derived from either hindpaw. The results constitute the first report of a lateralized deficit in opioid antinociception after unilateral inactivation of a specific brain area and show the first clear neuroanatomical dissociation between antinociceptive and motor effects of systemically administered morphine in the rat. The amygdala appears to modulate nociceptive signals entering the ipsilateral spinal dorsal horn, probably through monosynaptic connections with ipsilateral portions of the PAG.

Fig. 1.

Histological results of Experiment 1: unilateral Ce lesions, morphine and ipsilateral formalin. Representations of six coronal sections through the rat forebrain are shown in sequence from anterior to posterior. Thenumbers in the left margin indicate millimeters posterior to bregma. The closed curvesillustrate the borders of lesions that included the Ce (n = 9) in each hemisphere, as determined by the extent of neuronal cell loss and gliosis. Note that four rats had lesions placed in the left cerebral hemisphere, whereas the other five had lesions placed in the right cerebral hemisphere. The lesion area common to all rats in each hemisphere is shown as dark shading. Note that >95% of the Ce was damaged unilaterally in all nine rats. Adapted from Paxinos and Watson (1986). Amygdaloid areas: Ce, central nucleus; BLA, basolateral nucleus, anterior; BLV, basolateral nucleus, ventral; BSTIA, bed nucleus of the stria terminalis, intra-amygdaloid division; Me, medial nucleus;BM, basomedial nucleus; Co, cortical amygdaloid nuclei. Extra-amygdaloid areas: GP, globus pallidus; CP, caudate-putamen.

Fig. 2.

Histological results of Experiment 1: unilateral Ce lesions, morphine and contralateral formalin. Representations of six coronal sections through the rat forebrain are shown in sequence from anterior to posterior. The numbers in the left margin indicate millimeters posterior to bregma. Theclosed curves illustrate the borders of lesions that included the Ce (n = 9) in each hemisphere, as determined by the extent of neuronal cell loss and gliosis. Note that four rats had lesions placed in the left cerebral hemisphere, whereas the other five had lesions placed in the right cerebral hemisphere. The lesion area common to all rats in each hemisphere is shown asdark shading. Note that >95% of the Ce was damaged unilaterally in all nine rats. Adapted from Paxinos and Watson (1986). Amygdaloid areas: Ce, central nucleus;BLA, basolateral nucleus, anterior; BLV, basolateral nucleus, ventral; BSTIA, bed nucleus of the stria terminalis, intra-amygdaloid division; Me, medial nucleus; BM, basomedial nucleus; Co, cortical amygdaloid nuclei. Extra-amygdaloid areas: GP, globus pallidus; CP, caudate-putamen.

Fig. 3.

Average nociceptive and horizontal locomotor activity scores of unilateral Ce-treated rats in Experiment 1: unilateral Ce lesions, morphine and ipsilateral formalin. Error bars indicate SEM. In this experiment, unilateral Ce lesion rats treated with morphine received a formalin injection into the hindpawipsilateral to their Ce lesion. Ratingscale nociceptive scores are shown in A, flinch nociceptive scores are shown in B, and horizontal locomotor activity scores are shown in C. Note the similar pattern of results obtained with the rating scale and flinch-frequency methods of nociceptive scoring. There were no significant differences in baseline (i.e., systemic saline) nociceptive or horizontal locomotor activity scores at any time point between unilateral Ce lesion rats and unilateral Ce sham lesion rats (also see Fig. 5). In unilateral Ce sham lesion rats, morphine sulfate (6 mg/kg, s.c.) produced significant antinociception compared with saline at most time points. In unilateral Ce lesion rats, however, the ability of morphine to produce antinociception was severely impaired. This effect was dissociable from the effects of morphine on horizontal locomotor activity, because morphine-induced increases in horizontal locomotor activity were unaffected by unilateral Ce lesions (C). A, *p < 0.05; **p < 0.01, Tukey’s HSD test, compared with unilateral Ce sham lesion rats treated with systemic morphine;^Xp < 0.05;^XXp < 0.01, Tukey’s HSD test, compared with unilateral Ce lesion rats treated with systemic saline.B, *p < 0.05; **p < 0.01, Mann–Whitney U test, compared with unilateral Ce sham lesion rats treated with systemic morphine; ^Xp < 0.05, Wilcoxon signed-rank test, compared with unilateral Ce lesion rats treated with systemic saline.

Fig. 4.

Average nociceptive and horizontal locomotor activity scores of unilateral Ce-treated rats in Experiment 1: unilateral Ce lesions, morphine and contralateral formalin. Error bars indicate SEM. In this experiment, unilateral Ce lesion rats treated with morphine received a formalin injection in the hindpawcontralateral to their Ce lesion. Rating scale nociceptive scores are shown in A, flinch nociceptive scores areshown in B, and horizontal locomotor activity scores are shown in C. Note the similar pattern of results obtained with the rating scale and flinch-frequency methods of nociceptive scoring. There were no significant differences in baseline (i.e., systemic saline) nociceptive or horizontal locomotor activity scores at any time point between unilateral Ce lesion rats and unilateral Ce sham lesion rats (also see Fig. 5). In unilateral Ce sham lesion rats, morphine sulfate (6 mg/kg, s.c.) produced significant antinociception compared with saline at most time points. Unlike lesion rats that received morphine and ipsilateral formalin (Fig. 3), unilateral Ce lesions did not affect morphine-induced suppression of pain derived from the contralateral hindpaw.B, ^Xp < 0.05;^XXp < 0.01, Mann–Whitney Utest, compared with unilateral Ce sham lesion rats treated with systemic saline.

Fig. 5.

Average nociceptive and horizontal locomotor activity scores of unilateral Ce sham and unilateral Ce lesion rats treated with systemic saline in Experiment 1. Rating scale nociceptive scores are shown in A, flinch nociceptive scores are shown in B, and horizontal locomotor activity scores are shown in C. Error bars indicate SEM. Saline control data collected in Experiment 1 (see Figs. 3, 4) are presented in the samefigure to illustrate clearly that a unilateral Ce lesion did not affect baseline nociceptive responses when formalin was delivered to the hindpaw ipsilateral to the lesion.

Fig. 6.

Histological results of Experiment 2: unilateral BL lesions, morphine and ipsilateral formalin. Representations of six coronal sections through the rat forebrain are shown in sequence from anterior to posterior. The numbers in the left margin indicate millimeters posterior to bregma. Theclosed curves illustrate the borders of BL lesions (n = 7), as determined by the extent of neuronal cell loss and gliosis. The lesion area common to all rats in each hemisphere is shown as light shading. Note that four rats had lesions placed in the left cerebral hemisphere, whereas the other three had lesions placed in the right cerebral hemisphere. Adapted from Paxinos and Watson (1986). Amygdaloid areas:Ce, central nucleus; BLA, basolateral nucleus, anterior; BLV, basolateral nucleus, ventral;BSTIA, bed nucleus of the stria terminalis, intra-amygdaloid division; Me, medial nucleus;BM, basomedial nucleus; Co, cortical amygdaloid nuclei. Extra-amygdaloid areas: GP, globus pallidus; CP, caudate-putamen.

Fig. 7.

Histological results of Experiment 2: unilateral BL lesions, morphine and contralateral formalin. Representations of six coronal sections through the rat forebrain are shown in sequence from anterior to posterior. The numbers in the left margin indicate millimeters posterior to bregma. Theclosed curves illustrate the borders of BL lesions (n = 8), as determined by the extent of neuronal cell loss and gliosis. The lesion area common to all rats in each hemisphere is shown as light shading. Note that four rats had lesions placed in the left cerebral hemisphere, whereas the other four had lesions placed in the right cerebral hemisphere. Adapted from Paxinos and Watson (1986). Amygdaloid areas: Ce, central nucleus; BLA, basolateral nucleus, anterior;BLV, basolateral nucleus, ventral; BSTIA, bed nucleus of the stria terminalis, intra-amygdaloid division;Me, medial nucleus; BM, basomedial nucleus; Co, cortical amygdaloid nuclei. Extra-amygdaloid areas: GP, globus pallidus;CP,= caudate-putamen.

Fig. 8.

Combined histological results of Experiments 1 and 2 (unilateral Ce or unilateral BL lesions combined with morphine plusipsilateral formalin). The histological results depicted in Figures 1 and 6 are combined into a composite representation of the unilateral lesions that reduced morphine antinociception (lesions that included the Ce; see Fig. 3) and the unilateral lesions that failed to reduce morphine antinociception (BL lesions; see Fig. 9) when formalin was administered to the hindpaw ipsilateral to the lesion. Thenumbers in the left margin indicate millimeters posterior to bregma. The dark shading in each hemisphere indicates the lesion area common to rats in the Ce lesion group of Experiment 1. The lesion area common to rats in the BL lesion group of Experiment 2 is shown as light shadingin each hemisphere. Adapted from Paxinos and Watson (1986). Amygdaloid areas: Ce, central nucleus; BLA, basolateral nucleus, anterior; BLV, basolateral nucleus, ventral; BSTIA, bed nucleus of the stria terminalis, intra-amygdaloid division; Me, medial nucleus;BM, basomedial nucleus; Co,= cortical amygdaloid nuclei. Extra-amygdaloid areas: GP, globus pallidus; CP, caudate-putamen.

Fig. 9.

Average nociceptive and horizontal locomotor activity scores of unilateral BL-treated rats in Experiment 2: unilateral BL lesions, morphine and ipsilateral formalin. Error bars indicate SEM. In this experiment, unilateral BL lesion rats treated with morphine received a formalin injection in the hindpawipsilateral to their BL lesion. Ratingscale nociceptive scores are shown in A, flinch nociceptive scores are shown in B, and horizontal locomotor activity scores are shown in C. Note the similar pattern of results obtained with the rating scale and flinch-frequency methods of nociceptive scoring. There were no significant differences in baseline (i.e., systemic saline) nociceptive or horizontal locomotor activity scores at any time point between unilateral BL lesion rats and unilateral BL sham lesion rats. In unilateral BL sham lesion rats, morphine sulfate (6 mg/kg, s.c.) produced significant antinociception compared with saline at most time points. Unlike Ce lesion rats that received morphine and ipsilateral formalin (Fig. 3), unilateral BL lesions did not affect morphine-induced suppression of pain derived from the ipsilateral hindpaw.

Fig. 10.

Average nociceptive and horizontal locomotor activity scores of unilateral BL-treated rats in Experiment 2: unilateral BL lesions, morphine and contralateral formalin. Error bars indicate SEM. In this experiment, unilateral BL lesion rats treated with morphine received a formalin injection in the hindpawcontralateral to their BL lesion. Ratingscale nociceptive scores are shown in A, flinch nociceptive scores are shown in B, and horizontal locomotor activity scores are shown in C. Note the similar pattern of results obtained with the rating scale and flinch-frequency methods of nociceptive scoring. There were no significant differences in baseline (i.e., systemic saline) nociceptive or horizontal locomotor activity scores at any time point between unilateral BL lesion rats and unilateral BL sham lesion rats. In both unilateral BL sham lesion and unilateral BL lesion rats, morphine sulfate (6 mg/kg, s.c.) suppressed pain associated with injection of formalin into the contralateral hindpaw. A, *p < 0.01, Tukey’s HSD test, compared with unilateral BL sham lesion rats treated with systemic morphine.B, ^Xp < 0.05, Wilcoxon signed-rank test, compared with unilateral BL sham lesion rats treated with systemic saline.

Fig. 11.

Average nociceptive and horizontal locomotor activity scores of unilateral Ce-treated rats in Experiment 3. Error bars indicate SEM. Rating scale nociceptive scores are shown inA, flinch nociceptive scores are shown inB, and horizontal locomotor activity scores are shown inC. In this experiment, Ce-treated rats (sham lesion and lesion) received systemic morphine and intraplantar formalin on two separate occasions. On one occasion, formalin was delivered to the hindpaw ipsilateral to theCe treatment (lesion or sham lesion), and on the other occasion formalin was delivered to the contralateralhindpaw. Note the similar pattern of results obtained with the rating scale and flinch-frequency methods of nociceptive scoring. Under morphine, there were no significant differences in nociceptive scores between either group of unilateral Ce sham lesion rats and unilateral Ce lesion rats treated with contralateral formalin. In unilateral Ce lesion rats treated with ipsilateralformalin, however, morphine sulfate (6 mg/kg, s.c.) produced significantly less antinociception compared with all other groups. As was the case in Experiment 1 (Fig. 3), this effect was dissociable from the effects of morphine on horizontal locomotor activity, because morphine-induced increases in locomotor activity were unaffected by unilateral Ce lesions (C). B, *p < 0.05, Mann–Whitney U test, compared with all other groups.

Fig. 12.

Dose–effect relations for morphine in rats with unilateral Ce lesions or unilateral Ce sham lesions (n = 5–8 per group). For these curves, the dose of morphine was varied, whereas the concentration of formalin injected into either the ipsilateral (A) orcontralateral (B) hindpaw was held constant at 1.0%. Rating scale nociceptive scores were averaged across the second phase of the formalin test for all rats. The mean second phase nociceptive score for systemic saline-treated control rats (either lesion or sham lesion) was used asE_min for calculation of percentage of maximum possible antinociceptive effect (%MPE; see Experiment 4, Materials and methods).

Fig. 13.

Concentration–effect relations for morphine in rats with unilateral Ce lesions or unilateral Ce sham lesions (n = 6 per group). For these curves, the concentration of formalin injected into either theipsilateral (A) orcontralateral (B) hindpaw was varied, whereas the dose of morphine was held constant at 5 mg/kg. Rating scale nociceptive scores were averaged across the second phase of the formalin test for all rats. The mean second phase nociceptive score for systemic saline-treated control rats (either lesion or sham lesion) was used as E_min for calculation of percentage of maximum possible antinociceptive effect (%MPE; see Experiment 4, Materials and methods).

Fig. 14.

Histological results of Experiment 5 (unilateral muscimol injection into the Ce). Representations of three coronal sections through the rat amygdala are shown in sequence from anterior to posterior. The numbers in the left margin indicate millimeters posterior to bregma. Thefilled circles in each hemisphere show the approximate positions of the cannula tips corresponding to rats in the intra-Ce muscimol treatment group (n = 7). Adapted fromPaxinos and Watson (1986). Amygdaloid areas: Ce, central nucleus; BLA, basolateral nucleus, anterior;BLV, basolateral nucleus, ventral; BSTIA, bed nucleus of the stria terminalis, intra-amygdaloid division;Me, medial nucleus; BM, basomedial nucleus; Co, cortical amygdaloid nuclei. Extra-amygdaloid areas: GP, globus pallidus;CP,= caudate-putamen.

Fig. 15.

Average nociceptive scores of unilateral Ce-treated rats in Experiment 5. Rating scale nociceptive scores are shown in A and flinch nociceptive scores are shown inB. Error bars indicate SEM. In this experiment, Ce-treated rats received systemic morphine and intraplantar formalin on two separate occasions. On one occasion, formalin was delivered to the hindpaw ipsilateral to the unilateral Ce treatment (muscimol or saline), and on the other occasion formalin was delivered to the contralateral hindpaw. Note the similar pattern of results obtained with the rating scale and flinch-frequency methods of nociceptive scoring. There were no significant differences in nociceptive scores under morphine between either group of unilateral Ce sham lesion rats and unilateral Ce lesion rats treated withcontralateral formalin. In unilateral Ce lesion rats treated with ipsilateral formalin, however, morphine sulfate (4 mg/kg, s.c.) produced significantly less antinociception compared with allother groups. B, *p < 0.05; **p < 0.01, Mann–Whitney Utest, compared with both groups of unilateral Ce sham lesion rats treated with systemic morphine; ^Xp < 0.05;^XXp < 0.01, Wilcoxon signed-rank test, compared with unilateral Ce lesion rats treated with systemic morphine plus contralateral formalin.

Fig. 16.

Simplified diagram of endogenous pain control circuitry in the rat brain. The diagram emphasizes centers that contribute to the antinociceptive effect of systemically administered morphine, including two novel forebrain contributors, the amygdala and the posterior hypothalamic area (PHA; Manning et al., 1994; Manning and Franklin, 1998). The amygdala is interconnected with the PAG and PHA (see references in Manning and Franklin, 1998).A, amygdala; PAG, midbrain periaqueductal gray matter; RVM, rostral ventromedial medulla.