Acute noxious stimulation modifies morphine effect in serotonergic but not dopaminergic midbrain areas

Abstract

It is poorly understood if and how pain may modify the effect of opioids on neural systems that contribute to reward and addictive behavior. We hypothesized that the activation of ascending dopaminergic and serotonergic nuclei by morphine is modified by the presence of noxious stimulation. Immunohistochemical double-labeling technique with Fos was used to examine if an intraplantar formalin injection, an acute noxious input, changed the effect of morphine on dopaminergic neurons of the ventral tegmental area (VTA), and serotonergic neurons of the dorsal raphe nucleus (DR). Four groups of rats were analyzed: (1) control injected with normal saline s.c., (2) rats treated with formalin into the hind paw 30 min after normal saline injection, (3) rats injected with morphine sulfate s.c., and (4) rats treated with formalin into the hind paw 30 min after morphine injection (morphine/formalin). Following morphine injection, there was an increase in the number of dopaminergic neurons in the VTA with Fos immunolabeling. However, noxious stimulation did not detectably change morphine's effect on Fos expression in VTA dopamine neurons. In contrast, the number of serotonergic neurons containing Fos was increased in the morphine/formalin group compared to all other groups and this effect was topographically selective for the dorsal area of the DR at mid rostro-caudal levels. Therefore, morphine's activation of the VTA, which is associated with motivated behavior and reward seeking, appears similar in the context of pain. However, activation of the ascending serotonin system, which influences mood and has the capacity to modify reward pathways, appears different. In addition, these findings reveal interactions between nociceptive signaling and opioids that contrasts with the notion that opioids simply block access of nociceptive signaling to supraspinal structures.

Figure 1. Anatomical regions of analysis

Schematic representation of rat brain representative transverse brainstem sections modified from Paxinos Atlas (Paxinos and Watson, 1997). Panels A-D correspond to Paxinos Atlas plates 44, 49, 52, and 55, respectively. Anatomical areas of analysis are marked by square throughout the midbrain sections. Anatomical regions of interest include: tyrosine hydroxylase-immunoreactive dopaminergic neurons of the ventral tegmental area (VTA) (Panel A) and tryptophan hydroxylase-immunoreactive serotonergic neurons of the dorsal raphe nucleus (DR; Panel B - D). Please note that DR is divided into ROSTRAL (Panel B), MID (Panel C) and CAUDAL (Panel D) subgroups. Each of these subgroups is further divided into dorsal (DRD) and ventral part (DRV), and in the case of mid portion of the DR, also into the ventrolateral part (DRVL) that is located in the area of the ventrolateral periaqueductal gray (vlPAG) (Panel C). Numbers in the right upper corner represent distance from Bregma (in mm). Abbreviations: Aq, aqueduct (Sylvius); CnF, cuneiform nucleus; DRD, dorsal raphe nucleus, dorsal part; DRV, dorsal raphe nucleus, ventral part; DRVL, dorsal raphe nucleus, ventrolateral part; LSO, lateral superior olive; ml, medial lemniscus; PaR, pararubral nucleus; PnO, pontine reticular nucleus, oral part; py, pyramidal tract; scp, superior cerebellar peduncle; SNC, substantia nigra, compact part; SNR, substantia nigra, reticular part; tth, trigeminothalamic tract; vlPAG, ventrolateral periaqueductal gray; VTA, ventral tegmental area.

Figure 2. Estimated density of double-labeled monoamine neurons with Fos

Each graph represents biased estimates of density (# double-labeled cells/section/brain) ± SD of double-labeled immunoreactive monoamine neurons with Fos in different brain regions that include dopaminergic neurons of the ventral tegmental area (Panel 1), and serotonergic neurons of the dorsal raphe nucleus (DR; Panels 2-8). The latter is divided into three subgroups: rostral (Panels 2 and 3), mid (Panels 4-6), and caudal (Panels 7 and 8), which are then further divided into dorsal and ventral parts, and in the case of mid DR also into the ventrolateral part (Panel 6). Graphs are numbered and correspond to anatomical regions distributed from the most rostral to the most caudal midbrain regions (see Fig. 1 for schematic illustrations of matching anatomical regions of interest). Analysis included four different experimental groups: (1) control, (2) formalin, (3) morphine, and (4) morphine/formalin group (M/F). Statistically significant difference in estimated density among 4 different experimental groups is found in following anatomical regions of interest: VTA (F(3,20)=14.625; p<0.001; Panel 1), dorsal part of rostral DR (F(3,20)=5.568; p=0.006; Panel 2), dorsal part of the mid DR (F(3,20)=5.411; p=0.007; Panel 4), ventral part of the mid DR (F(3,20)=6.261; p=0.004; Panel 5), and ventrolateral part of the mid DR (F(3,20)=3.776; p=0.027; Panel 6). Two-tailed p value less than 0.05 with Tukey correction was considered statistically significant. Double asterisks (**) denote statistically significant difference from control and formalin groups only, whereas single asterisk (*) denotes statistically significant difference in values from control, formalin, as well as morphine treatment groups. More specifically, in the VTA (Panel 1), pairwise comparisons with Tukey corrections of all groups showed statistically significant increase in density of double-labeled neurons in the morphine group compared to control (p<0.001) and formalin (p<0.001), and M/F group compared to control (p=0.011) and formalin (p=0.008). However, morphine and M/F groups were not statistically different between each other (p=0.287) in the VTA. Note that in the DR, morphine treatment did not cause any change in density of double-labeled serotonergic neurons with Fos. Furtheromore, in the DR, pairwise comparisons with Tukey corrections of experimental groups showed statistically significant increase in estimated density of double-labeled neurons in the morphine/formalin group compared to control (p=0.026), formalin (p=0.008), as well as morphine group (p=0.042) only in the dorsal area of the mid DR (Panel 4). In addition, statistically significant increase in density of double-labeled serotonergic neurons in morphine/formalin treatment group in comparison to control and formalin but not morphine treatment groups, were found in dorsal part of rostral DR (p=0.010 versus control; p=0.011 versus formalin; Panel 2), ventral part of the mid DR (p=0.006 versus control; p=0.007 versus formalin; Panel 5), and ventrolateral part of the mid DR (p=0.049 versus control; p=0.042 versus formalin; Panel 6). Finally, the estimated density of serotonergic neurons that were double-labeled with Fos showed no statistically significant difference among the four treatment groups in the following subgregions of the DR: ventral part of the rostral DR (F(3,20)=2.337; p=0.104; Panel 3), dorsal part of the caudal DR (F(3,20)=0.709; p=0.559; Panel 7), and ventral part of the caudal DR (F(3,20)=1.126; p=0.362; Panel 8).

Figure 3. Immunohistochemical labeling of dopaminergic neurons in the ventral tegmental area

Fos immunolabeling (red) and tyrosine hydroxylase containing dopaminergic neurons (green) of the ventral tegmental area in four different experimental groups: (A) Control, (B) Formalin, (C) Morphine, and (D) Morphine/Formalin group (M/F). See Fig. 1A for schematic anatomical location of ventral tegmental area distribution. Arrowheads illustrate Fos immunolabeling only. Note the increased number of double-labeled neurons that are depicted both in morphine (C) and M/F group (D) as illustrated by arrows. No significant differences in number of double-labeled neurons were found between morphine (C) and M/F (D) treatment groups (see also Fig. 2, Panel 1 for statistical analysis). Scale bar = 100 μm throughout.

Figure 4. Immunohistochemical labeling of serotonergic neurons in the dorsal raphe nucleus

Panels A-D represent area of the mid dorsal raphe nucleus, whereas Panels A′-D′ illustrate ventrolateral part of the mid nucleus raphe magnus located in the ventrolateral periaqueductal gray. See Fig. 1C for schematic anatomical location of dorsal and ventrolateral part of the mid dorsal raphe nucleus. Fos immunolabeling (red) and tryptophan hydroxylase (TPOH) containing serotonergic neurons (green) of the dorsal raphe nucleus are shown in four different experimental groups: (A, A′) Control, (B, B′) Formalin, (C, C′) Morphine, and (D, D′) Morphine/Formalin group (M/F). Note individual nuclear Fos immunolabeling in each of the treatment groups as indicated by arrowheads. In control rats (A′), occasional TPOH-immunoreactive serotonergic neuron was double-labeled with Fos (arrow). In morphine injected rats (C, C′), some TPOH-immunoreactive serotonergic neurons were double-labeled with Fos (arrows). Note the increased number of double-labeled neurons that are depicted in M/F group (D, D′) as illustrated by arrows (see also Fig. 2, Panel 4 and 6 for statistical analysis). Scale bar = 100 μm throughout.