Neurobiological Correlates of Pain Avoidance-Like Behavior in Morphine-Dependent and Non-Dependent Rats

Abstract

Repeated use of opioids can lead to the development of analgesic tolerance and dependence. Additionally, chronic opioid exposure can cause a paradoxical emergence of heightened pain sensitivity to noxious stimuli, termed hyperalgesia, which may drive continued or escalated use of opioids to manage worsening pain symptoms. Opioid-induced hyperalgesia has traditionally been measured in rodents via reflex-based assays, including the von Frey method. To better model the cognitive/motivational dimension of pain in a state of opioid dependence and withdrawal, we employed a recently developed non-reflex-based method for measuring pain avoidance-like behavior in animals (mechanical conflict avoidance test). Adult male Wistar rats were administered an escalating dose regimen of morphine (opioid-dependent group) or repeated saline (control group). Morphine-dependent rats exhibited significantly greater avoidance of noxious stimuli during withdrawal. We next investigated individual relationships between pain avoidance-like behavior and alterations in protein phosphorylation in central motivation-related brain areas. We discovered that pain avoidance-like behavior was significantly correlated with alterations in phosphorylation status of protein kinases (ERK, CaMKII), transcription factors (CREB), presynaptic markers of neurotransmitter release (Synapsin), and the rate-limiting enzyme for dopamine synthesis (TH) across specific brain regions. Our findings suggest that alterations in phosphorylation events in specific brain centers may support cognitive/motivational responses to avoid pain.

Keywords: avoidance; hippocampus; opioid dependence; pain; prefrontal cortex; striatum.

Figure 1

The mechanical conflict-avoidance task is a non-reflex-based method that was employed to model increases in the motivation to avoid pain during opioid withdrawal. The mechanical conflict-avoidance apparatus contains three red acrylic chambers: a brightly lit (aversive) start chamber, a probe chamber of adjustable probes heights (0 mm, 0.5 mm, 1 mm, 2 mm, 3 mm, 4 mm, 5 mm), and a dark (rewarding) goal chamber. Training consisted of rats being given a choice between remaining in a lighted box or crossing over elevated probes (noxious stimulus) to reach a goal box. In this model, a longer latency to exit onto the probes is thought to reflect an increased motivation to avoid pain.

Figure 2

(A) Experimental timeline. After initial training, animals were given 7 days of mechanical conflict-avoidance testing to assess baseline measurement of avoidance and to split rats into two equivalent groups. Rats were given two weeks of either escalating morphine (week 1-10 mg/kg, week 2-20 mg/kg) or saline injections every 24 hours to induce opioid dependence. Tests of mechanical hypersensitivity were conducted at the end of week 1 and week 2. To assess mechanical conflict-avoidance in morphine-dependent rats compared to saline controls, the stimulus-response assessment was repeated after two weeks of daily injections. During testing, rats received an additional week of either morphine (20 mg/kg) or saline injections. Behavioral measurements for each day were taken 24 hours after the previous drug injection (acute withdrawal in morphine-dependent rats). All animals were euthanized 24 hours after the final drug injection and brains were immediately dissected and snap-frozen in preparation for regional tissue sample collection. (B-D) Schematic representation of sub-regional brain samples collected (Paxinos and Watson, 1998). (B) DM = dorsomedial prefrontal cortex; (B) VM = ventromedial prefrontal; (C) DS = dorsal striatum; (C) VS = ventral striatum; (D) HIP = hippocampus.

Figure 3

Von Frey testing. Rats were given two weeks of either escalating morphine (week 1: 10 mg/kg, week 2: 20 mg/kg) or saline injections every 24 hours to induce opioid dependence. Tests of mechanical hypersensitivity (von Frey) were conducted at the end of week 1 and week 2. In morphine-dependent animals in acute withdrawal, there was a significant decrease in von Frey thresholds between weeks 1 and 2 in (A) experimental group 1 (*p<0.05) and (B) experimental group 2 (**p<0.01), indicating heightened mechanical hypersensitivity. In saline controls, there was no difference in the von Frey thresholds between weeks 1 and 2 in (C) experimental group 1 (p>0.05) and (D) experimental group 2 (p>0.05).

Figure 4

Mechanical conflict-avoidance testing. Rats were given two weeks of injections of either an escalating dose regimen of morphine (to induce opioid dependence) or saline. Rats were then given a choice between remaining in a lighted chamber or crossing over elevated probes to reach a goal chamber. There was a longer latency to exit onto the probes in morphine-dependent rats compared to controls (*p<0.05, ***p<0.0001 main effect of group across all probe heights; ###p<0.0001 main effect of probe heights) in experimental group 1 (A), group 2 (B), and groups 1 & 2 combined (C). (D) Changes in mechanical sensitivity (Von Frey test) over the week prior to the mechanical conflict avoidance testing negatively correlated with individual levels of subsequent pain avoidance-like behavior (r=−.4413; p<0.05). Black dots represent morphine-dependent rats in 24-hour withdrawal, while grey dots represent saline controls.

Figure 5

Neurobiological correlates of pain avoidance-like behavior in the dorsomedial prefrontal cortex (dmPFC). (A) Pain avoidance was negatively correlated with phosphorylation of Synapsin^S9 in the dmPFC (r=−0.7569; p<0.05). (B) Pain avoidance was negatively correlated with phosphorylation of CREB^S133 in the dmPFC (r=−0.6493; p<0.05). Black dots represent morphine-dependent rats in 24-hour withdrawal, while grey dots represent saline controls.

Figure 6

Neurobiological correlates of pain avoidance-like behavior in the ventromedial prefrontal cortex (vmPFC). (A) Pain avoidance was positively correlated with phosphorylation of Synapsin^S9 in the vmPFC (r=0.7645; p<0.05). (B) Pain avoidance was positively correlated with phosphorylation of CaMKII^Thr286 in the vmPFC (r=0.6835; p<0.05). Black dots represent morphine-dependent rats in 24-hour withdrawal, while grey dots represent saline controls.

Figure 7

Neurobiological correlates of pain avoidance-like behavior in the striatum. (A) Pain avoidance was positively correlated with phosphorylation of TH^S40 in the ventral striatum (r=0.7495; p<0.05). (B) Pain avoidance was negatively correlated with phosphorylation of CaMKII^Thr286 in the ventral striatum (r=−0.7881; p<0.05). (C) Pain avoidance was negatively correlated with phosphorylation of TH^S40 in the dorsal striatum (r=−0.6774; p<0.05). Black dots represent morphine-dependent rats in 24-hour withdrawal, while grey dots represent saline controls.

Figure 8

Neurobiological correlates of pain avoidance-like behavior in the hippocampus. Pain avoidance was positively correlated with phosphorylation of ERK in the dorsal hippocampus (r=0.7223; p<0.05). Black dots represent morphine-dependent rats in 24-hour withdrawal, while grey dots represent saline controls.

Figure 9

Summary of observed neurobiological correlates of pain avoidance-like behavior. Our findings suggest that increased pain avoidance is associated with decreased pre-synaptic transmission capacity (pSynapsin) and a subsequent reduction in post-synaptic activation of CREB in the dmPFC. In contrast, increased pain avoidance is associated with increased pre-synaptic vesicle release (pSynapsin) and a subsequent increase in post-synaptic activation of CaMKII in the vmPFC. Weakened dmPFC activity disinhibits downstream stress responses, while strengthened vmPFC activity facilitates the stress response. Accordingly, increases in PFC-mediated stress signaling may facilitate pain sensitivity on the mechanical conflict-avoidance task. Our findings suggest that increased pain avoidance is also associated with lower levels of dopamine in the VS (as reflected by less feedback inhibition on pTH⁴⁰ and decreased activation of CaMKII), but higher levels of dopamine in the DS (as reflected by greater feedback inhibition of pTH⁴⁰), indicating that regulation of striatal dopamine signaling is closely associated with pain avoidance. Finally, increased pain avoidance is associated with increased phosphorylation of ERK in the dorsal hippocampus. Since ERK activity is necessary for the development and expression of negative affect-conditioned states, activation of ERK may dictate appropriate contextual responses to avoid noxious stimuli during the mechanical conflict-avoidance task.