Differences in carbachol dose, pain condition, and sex following lateral hypothalamic stimulation

Abstract

Lateral hypothalamic (LH) stimulation produces antinociception in female rats in acute, nociceptive pain. Whether this effect occurs in neuropathic pain or whether male-female sex differences exist is unknown. We examined the effect of LH stimulation in male and female rats using conditions of nociceptive and neuropathic pain. Neuropathic groups received chronic constriction injury (CCI) to induce thermal hyperalgesia, a sign of neuropathic pain. Nociceptive rats were naive for CCI, but received the same thermal stimulus following LH stimulation. To demonstrate that CCI ligation produced thermal hyperalgesia, males and females received either ligation or sham surgery for control. Both males and females demonstrated significant thermal hyperalgesia following CCI ligation (p<0.05), but male sham surgery rats also showed a significant left-right difference not present in female sham rats. In the second experiment, rats randomly assigned to CCI or nociceptive groups were given one of three doses of the cholinergic agonist carbachol (125, 250, or 500 nmol) or normal saline for control, microinjected into the left LH. Paw withdrawal from a thermal stimulus (paw withdrawal latency; PWL) was measured every 5 min for 45 min. Linear mixed models analysis showed that males and females in both pain conditions demonstrated significant antinociception, with the 500-nmol dose producing the greatest effect across groups compared with controls for the left paw (p<0.05). Female CCI rats showed equivalent responses to the three doses, while male CCI rats showed more variability for dose. However, nociceptive females responded only to the 500-nmol dose, while nociceptive males responded to all doses (p<0.05). For right PWL, only nociceptive males showed a significant carbachol dose response. These findings are suggestive that LH stimulation produces antinociception in male and female rats in both nociceptive and neuropathic pain, but dose response differences exist based on sex and pain condition.

Keywords: analgesia; antinociception; lateral hypothalamus; nociception; sex differences.

Figure 1

Representative location of microinjection sites in the LH for left PWL for the 500 nmol dose of carbachol in CCI rats. Most of the injection sites were located within the border of the LH between AP −2.12 and −3.60 mm from bregma. The symbols represent the differences between baseline paw response latencies and those at the peak time of carbachol effectiveness (10 min for females and 20 min for males). Symbols for response latency after microinjection of carbachol are as follows: (◇) 3–8 sec; (○) 9–14 sec; (□) 15–20 sec. Injection sites located in the zona incerta and amygdala (●;■) and the ventral thalamus or optic tract (◆) were excluded from data analysis. AH, anterior hypothalamic area; AMG, amygdala; ic, internal capsule; LH, lateral hypothalamus; LV, lateral ventricle; mt, mammillothalamic tract; opt, optic tract; PH, posterior hypothalamus; VM, ventromedial thalamic nucleus; VPL, ventral posterolateral thalamic nucleus; VPM; ventral posteromedial thalamic nucleus; ZI, zona incerta.

Figure 2

CCI ligation produced decreases in left PWL. (A) Female CCI rats (○) had significantly shorter paw withdrawal latencies than sham surgery controls (●). (B) Male CCI rats also had significantly shorter withdrawal latencies (□) compared to sham controls (■). (C) Both female and male CCI rats demonstrated shorter average PWL on the left (L) compared to the right (R) paw. However, while female sham surgery control rats showed no left/right paw differences, males sham surgery controls had significantly shorter left PWL compared to right paws (averaged response latencies at 10 min post baseline). Mean response latencies ± SEM plotted on the ordinate as a function of time; n = 14 per group (*p < 0.05).

Figure 3

Microinjection of carbachol in the left LH produced dose-dependent increases in left PWL. Following a baseline response latency measurement at time −1 min, normal saline (◆) or carbachol in one of three doses: 125 nmol (△); 250 nmol (□); or 500 nmol (○) was microinjected into the LH at time 0. The 500 nmol dose was the most effective across groups compared to saline control rats, but dose response was dependent on sex of the rat and pain condition. Female CCI rats (A) showed equivalent responses to the three carbachol doses, while male CCI rats (B) showed more variability for dose and a smaller, but significant, response to LH stimulation than females. However, nociceptive females responded only to the 500 nmol dose (C), while nociceptive males responded to all carbachol doses (D). Mean response latencies ± SEM are plotted on the ordinate as a function of time (min).

Figure 4

Effects of carbachol microinjection in the left LH on right PWLs. Neither CCI group (A and B) showed a significant right paw effect at any dose of carbachol (125 nmol (△); 250 nmol (□); or 500 nmol (○), compared to saline for control (◆), nor did female nociceptive rats (C). Male nociceptive rats (D) demonstrated a significant increase in PWL at all doses of carbachol compared to the saline control group. Mean response latencies ± SEM plotted on the ordinate as a function of time (min).