Differential effects of neurotoxic destruction of descending noradrenergic pathways on acute and persistent nociceptive processing.

Although many pharmacological studies indicate that bulbospinal noradrenergic projections contribute to antinociception, lesions of the major brainstem noradrenergic cell groups have provided conflicting evidence. Here we used a new immunotoxin, anti-dopamine beta-hydroxylase-saporin, to re-examine the contribution of noradrenergic pathways to nociception and to morphine analgesia. We treated rats intrathecally by lumbar puncture with the immunotoxin and examined dopamine beta-hydroxylase (DbetaH) immunoreactivity seven and 14 days after treatment. There was no change in DbetaH staining at 7 days; however, 14 days after treatment we demonstrated significant destruction of noradrenergic neurons in the locus coeruleus and in the A5 and A7 cell groups. There was a concomitant loss of noradrenergic axons in the dorsal and ventral horns of the lumbosacral and cervical cord. Consistent with the lack of anatomical changes, we found no difference in nociceptive responses in the hot-plate, tail-flick or formalin tests one week post-toxin. On day 14 we examined the behavioral response to injection of formalin into the hindpaw and found that responses during the second phase of pain behavior were significantly reduced. There was no change during the first phase. Formalin-evoked fos expression in the spinal cord was also reduced. We also evaluated morphine analgesia in the formalin test and found that toxin-treated animals exhibited enhanced morphine analgesia. These results establish the utility of using this immunotoxin to selectively destroy subpopulations of noradrenergic cell groups and provide evidence that acute and persistent nociception are differentially regulated by descending noradrenergic pathways.

Activation of coeruleospinal noradrenergic inhibitory controls during withdrawal from morphine in the rat.

We previously reported that withdrawal from morphine induces the expression of Fos, a marker of neuronal activity, in spinal cord neurons, particularly in laminae I and II of the superficial dorsal horn, and that the magnitude of Fos expression is increased in rats with a midthoracic spinal transection. We suggested that loss of withdrawal-associated increases in descending inhibitory controls that arise in the brainstem underlie the increased Fos expression after spinal transection. Here, we addressed the origin of the supraspinal inhibition. We injected rats intracerebroventricularly with saline or anti-dopamine-beta-hydroxylase-saporin, a toxin that destroys noradrenergic neurons of the locus coeruleus. Eleven days later, we implanted rats with morphine or placebo pellets, and after 4 d, we precipitated withdrawal with naltrexone. One hour later, the rats were killed, their brains and spinal cords were removed, and transverse sections of the brains and spinal cords were immunoreacted with an antibody to Fos. In placebo-pelleted rats, the toxin injection did not alter behavior and did not induce expression of the Fos protein. However, compared with saline-injected withdrawing rats, the toxin-treated rats that underwent withdrawal demonstrated an intense withdrawal behavior rarely seen in the absence of toxin, namely forepaw fluttering. The rats also had significantly increased Fos-like immunoreactivity in all laminae of the cervical cord and in laminae I and II and the ventral horn of the lumbar cord. No differences were recorded in the sacral cord. We conclude that the effects of spinal transection in rats that withdraw from morphine in part reflect a loss of coeruleospinal noradrenergic inhibitory controls.

The contribution of supraspinal, peripheral and intrinsic spinal circuits to the pattern and magnitude of Fos-like immunoreactivity in the lumbar spinal cord of the rat withdrawing from morphine.

Withdrawal from morphine evokes increases in Fos-like immunoreactivity in the spinal cord, particularly in the superficial dorsal horn, laminae I/II. To determine the origin of the increased Fos-like immunoreactivity, we selectively targeted central or peripheral opioid receptors with naloxone-methiodide, an antagonist that does not cross the blood-brain barrier, or induced withdrawal after eliminating possible sources of input to the superficial dorsal horn. To induce tolerance, we implanted rats with morphine or placebo pellets (75 mg, six pellets over three days). On day 4, withdrawal was precipitated and after 1 h, the rats were killed, their spinal cords removed and 50 microm transverse sections of the spinal cord immunoreacted with a rabbit polyclonal antiserum directed against the Fos protein. In placebo-pelleted rats, none of the different procedures, viz. spinal transection, unilateral dorsal rhizotomy (L4-S2), neonatal capsaicin treatment or direct intrathecal opioid antagonist injection, induced expression of the Fos protein. However, both spinally transected and rhizotomized withdrawing animals showed significant increases in Fos-like immunoreactivity in laminae I/II, compared to intact withdrawing rats. Neonatal treatment with capsaicin, which eliminates C-fibres, did not alter Fos-like-immunoreactivity. Selective withdrawal of morphine from peripheral opioid receptors by naloxone-methiodide did not induce Fos-like immunoreactivity in the lumbar spinal cord greater than that recorded in nonwithdrawing rats. However, intrathecal injection of naloxone-methiodide increased Fos-like immunoreactivity in laminae I/II and the ventral horn to a greater extent than did subcutaneous injection of naloxone. We hypothesize that the increased Fos expression after systemic withdrawal in spinally-transected rats results from a loss of descending inhibitory control that is activated during withdrawal. The increase in withdrawal-induced Fos-like immunoreactivity after rhizotomy may be secondary to loss of inhibitory controls exerted by large diameter primary afferents or to deafferentation-induced reorganization in the dorsal horn. Since capsaicin did not alter the magnitude of Fos-like immunoreactivity in withdrawing rats, we conclude that hyperactivity of opioid receptor-laden C-fibres is not a necessary contributor to the withdrawal-induced increase in Fos-like immunoreactivity in laminae I and II. Taken together with the results recorded after intrathecal injection of naloxone-methiodide in tolerant rats, we conclude that the pattern of lumbar spinal cord Fos expression following systemic withdrawal is primarily a consequence of increased activity in opioid receptor-containing circuits intrinsic to the dorsal horn and that the magnitude of Fos expression is normally dampened by supraspinal and primary afferent-derived inhibitory inputs.

Formalin-evoked Fos expression in spinal cord is enhanced in morphine-tolerant rats.

It has been hypothesized that tolerance to the analgesic effects of morphine results from the development of a compensatory response in neurons that express the opioid receptor or in neural circuits in which those neurons participate. The compensatory response establishes a sensitized state in these neurons. To determine if administration of a noxious stimulus can unmask a sensitization of dorsal horn neurons in morphine-pelleted rats, we injected morphine-tolerant and control rats with formalin into the plantar surface of the hindpaw, counted the number of flinches for 2 h and then processed the lumbar cord for Fos immunocytochemistry. Although there was no significant difference in flinching behavior between the morphine-tolerant and control groups, we recorded significantly increased total Fos-like immunoreactivity at the L4/5 and L2 segments both ipsilateral and contralateral to the site of formalin injection in the morphine-tolerant rats compared to the control rats. These results suggest that lumbar spinal cord neurons are sensitized during the development of tolerance, that the sensitization can be unmasked by the administration of a noxious stimulus and that it is manifested as increased expression of the Fos protein in the lumbar cord.

The differential contribution of capsaicin-sensitive afferents to behavioral and cardiovascular measures of brief and persistent nociception and to Fos expression in the formalin test.

Intraplantar injection of dilute formalin evokes brief (Phase 1) and persistent (Phase 2) increases in primary afferent activity, pain behavior, and cardiovascular responses, and induces spinal cord Fos-like immunoreactivity (Fos-LI). Although previous studies demonstrated that the destruction of small diameter primary afferents with neonatal capsaicin treatment decrease formalin-evoked nociception, these studies only evaluated behavioral responses, and did not distinguish between Phase 1 and 2. To address these questions, we simultaneously evaluated formalin-evoked pain behavior (flinching of the afflicted paw), cardiovascular responses (heart rate and mean arterial pressure), and lumbar spinal cord Fos expression in control rats and in rats treated with capsaicin (100 mg/kg) one day postpartum. We found that neonatal capsaicin-treated rats, compared to controls, exhibited similar cardiovascular responses and slightly less flinching behavior during Phase 1. During Phase 2, however, capsaicin-treated rats exhibited 59% less flinching and 45% smaller heart rate responses. Also, in capsaicin-treated rats, we counted 59% fewer Fos-labeled neurons in the spinal cord. These results indicate that capsaicin-sensitive afferents contribute to formalin-evoked behavioral and cardiovascular responses and to spinal cord neuronal responses. The differential effect of neonatal capsaicin on nociception during Phase 1 and Phase 2 suggests that sensitization mechanisms during Phase 1 do not contribute to the magnitude of nociceptive responses during Phase 2.

Contribution of sacral spinal cord neurons to the autonomic and somatic consequences of withdrawal from morphine in the rat.

In this study, we monitored Fos-like immunoreactivity in the sacral spinal cord to identify neurons that are likely to contribute to the autonomic manifestations of opioid antagonist-precipitated withdrawal in morphine-tolerant rats. Injection of systemic antagonist increased the Fos-like immunoreactivity throughout the first sacral segment, particularly in laminae I/II, X, and in the sacral parasympathetic nucleus (SPN). Selective peripheral withdrawal, with a hydrophilic antagonist that does not cross the blood-brain barrier (BBB), induced diarrhea, but no other withdrawal signs were evident. Compared to rats that withdrew systemically, peripherally withdrawal evoked significantly less Fos-like immunoreactivity in laminae V/VI, X and the SPN. By contrast, selective spinal withdrawal, by intrathecal injection of an opioid antagonist that does not cross the BBB, provoked hyperactivity of the hindlimbs and tail, but no diarrhea. These animals demonstrated significantly increased Fos-like immunoreactivity in laminae I/II, V/VI, the SPN, and the ventral horn compared to rats that withdrew systemically. Animals treated neonatally with capsaicin, to eliminate C-fiber input, demonstrated withdrawal behavior similar to intact withdrawing rats, except that the capsaicin-pretreated rats had significantly greater weight loss. However, this group had less Fos-like immunoreactivity in laminae V/VI, X and SPN compared to the intact withdrawing rats. These data suggest that withdrawal from morphine evokes hyperactivity of sacral neurons, particularly those involved in regions that process nociceptive and autonomic information. Peripheral withdrawal is sufficient to induce diarrhea, but it does not fully explain the associated weight loss. Unmyelinated primary afferents may contribute a tonic peripheral inhibition of circuits that regulate gut motility and intestinal fluid transport. Taken together, these data suggest that chronic exposure to opioids induces a latent sensitization in sacral cord neurons that can be manifested as neuronal hyperactivity during withdrawal; this mechanism may underlie withdrawal-induced hyperalgesia and gut hypermotility.

Spinal cord mechanisms of opioid tolerance and dependence: Fos-like immunoreactivity increases in subpopulations of spinal cord neurons during withdrawal [corrected].

Tolerance to the analgesic effects of morphine results in part from the development of a compensatory response in neurons that express the opioid receptor or of neural circuits in which those neurons participate. According to this formulation, withdrawal of morphine results in an overshoot of several neuronal properties because of the unopposed action of the compensatory response system. To identify the population of spinal cord neurons that underlies this state, we monitored expression of Fos-like immunoreactivity, after naltrexone-precipitated abstinence in normal and morphine-tolerant rats. After daily (five days) implantation of morphine or placebo pellets, the rats received an injection of saline or naltrexone and behavior was monitored for 1 h. The rats were then killed, their spinal cords removed and 50-microns transverse sections of the lumbar cord were immunostained with a rabbit polyclonal antiserum directed against Fos. Naltrexone injection in the placebo group did not increase spinal cord Fos expression. Naltrexone-precipitated abstinence resulted in an increase in Fos expression at all levels of the spinal cord; the greatest increase and densest staining was in laminae I through VI. Importantly, when withdrawal was precipitated in anesthetized rats, we recorded a significant reduction in Fos expression, particularly in laminae III through VI, but there was persistent expression in the superficial dorsal horn, particularly in lamina I. These results suggest that spinal cord nociresponsive neurons are sensitized during the development of tolerance. This sensitization is unmasked by the administration of naltrexone and is manifested by fos induction in laminae I/II in awake or anesthetized withdrawing animals. The underlying mechanisms of tolerance development may be similar to those that underlie injury-induced central sensitization and hyperalgesia.