Spinal Dyn Research Articles

Postnatal maturation of spinal dynorphin circuits and their role in somatosensation.

Inhibitory interneurons in the adult spinal dorsal horn (DH) can be neurochemically classified into subpopulations that regulate distinct somatosensory modalities. Although inhibitory networks in the rodent DH undergo dramatic remodeling over the first weeks of life, little is known about the maturation of identified classes of GABAergic interneurons, or whether their role in somatosensation shifts during development. We investigated age-dependent changes in the connectivity and function of prodynorphin (DYN)-lineage neurons in the mouse DH that suppress mechanosensation and itch during adulthood. In vitro patch clamp recordings revealed a developmental increase in primary afferent drive to DYN interneurons and a transition from exclusive C-fiber monosynaptic input to mixed A-fiber and C-fiber innervation. Although most adult DYN interneurons exhibited tonic firing as expected from their inhibitory phenotype, neonatal and adolescent DYN cells were predominantly classified as phasic or single-spiking. Importantly, we also found that most of the inhibitory presynaptic terminals contacting lamina I spinoparabrachial projection neurons (PNs) originate from DYN neurons. Furthermore, inhibitory synaptic input from DYN interneurons onto PNs was weaker during the neonatal period, likely reflecting a lower number of GABAergic terminals and a reduced probability of GABA release compared to adults. Finally, spinal DYN interneurons attenuated mechanical sensitivity throughout development, but this population dampened acute nonhistaminergic itch only during adulthood. Collectively, these findings suggest that the spinal "gates" controlling sensory transmission to the brain may emerge in a modality-selective manner during early life due to the postnatal tuning of inhibitory synaptic circuits within the DH.

Blockade of non-opioid excitatory effects of spinal dynorphin A at bradykinin receptors.

Dynorphin A (Dyn A) is an endogenous opioid ligand that possesses neuroinhibitory (antinociceptive) effects via μ, δ, and κ opioid receptors. However, under chronic pain conditions, up-regulated spinal Dyn A can also interact with bradykinin receptors (BRs) to promote hyperalgesia through a neuroexcitatory(pronociceptive) effect. These excitatory effects cannot be blocked by an opioid antagonist, and thus are non-opioid in nature. On the basis of the structural dissimilarity between Dyn A and endogenous BR ligands, bradykinin(BK) and kallidin (KD), Dyn A's interaction with BRs could not be predicted, and provided an opportunity to identify a novel potential neuroexcitatory target. Systematic structure-activity relationship (SAR) studies discovered a minimum pharmacophore of Dyn A, [des-Arg7]-Dyn A-(4-11) LYS1044 for antagonist activity at the BRs, along with insights into the key structural features for BRs recognition, i.e., amphipathicity. The des-Tyr fragment of dynorphin does not bind to opioid receptors. Intrathecal administration of des-Tyr dynorphin produces hyperalgesia reminiscent of behaviors seen in peripheral n europathic pain models and at higher doses, neurotoxicity. Our lead ligand LYS1044 negatively modulated Dyn A-(2-13)-induced neuroexcitatory effects in naïve animals and blocked mechanical hypersensitivity and thermal hyperalgesia in a dose-dependent manner in animals with experimental neuropathic pain. Based on these results, ligand LYS1044 might prevent abnormal pain states by blocking the neuroexcitatory effects of increased levels of Dyn A that are seen in experimental models of neuropathic pain and that likely promote excitation mediated by BRs in the spinal cord.

Sex, pain, and opioids: interdependent influences of sex and pain modality on dynorphin-mediated antinociception in rats.

The role of dynorphin A (1-17; Dyn) and its associated kappa opioid receptor (KOR) in nociception represents a longstanding scientific conundrum: Dyn and KOR (Dyn/KOR) have variously been reported to inhibit, facilitate, or have no effect on pain. We investigated whether interactions between sex and pain type (which are usually ignored) influenced Dyn/KOR-mediated antinociception. Blockade of the spinal α(2)-noradrenergic receptor (α(2)-NAR) using yohimbine elicited comparable spinal Dyn release in females and males. Nevertheless, the yohimbine-induced antinociception exhibited sexual dimorphism that depended on the pain test used: in the intraperitoneal acetic acid-induced writhing test, yohimbine produced antinociception only in females, whereas in the intraplantar formalin-induced paw flinch test, antinociception was observed only in males. In females and males, both intrathecal Dyn antibodies and spinal KOR blockade eliminated the yohimbine-induced antinociception, indicating that Dyn/KOR mediated it. However, despite the conditional nature of spinal Dyn/KOR-mediated yohimbine antinociception, both intraplantar formalin and intraperitoneal acetic acid activated spinal Dyn neurons that expressed α(2)-NARs. Moreover, Dyn terminals apposed KOR-expressing spinal nociceptive neurons in both sexes. This similar organization suggests that the sexually dimorphic interdependent effects of sex and pain type may result from the presence of nonfunctional (silent) KORs on nociceptive spinal neurons that are responsive to intraplantar formalin (in females) versus intraperitoneal acetic acid (in males). Our findings that spinal Dyn/KOR-mediated antinociception depends on interactions between sex and pain type underscore the importance of using both sexes and multiple pain models when investigating Dyn/KOR antinociception.

Regulation of Spinal Dynorphin 1-17 Release by Endogenous Pituitary Adenylyl Cyclase-Activating Polypeptide in the Male Rat: Relevance of Excitation via Disinhibition

Opioids inhibit release of primary afferent transmitters but it is unclear whether the converse occurs. To test the hypothesis that primary afferent transmitters influence opioid-ergic tone, we studied the functional and anatomical relationships between pituitary adenylyl cyclase-activating polypeptide (PACAP) and dynorphin 1-17 (Dyn) in spinal cord. We found that activation of the PACAP-specific receptor PAC(1) (PAC(1)R) inhibited, whereas PAC(1)R blockade augmented, spinal release of Dyn. It is noteworthy that in the formalin-induced pain model PAC(1)R blockade (via PACAP6-38) also resulted in antinociception that was abolished by spinal κ-opioid receptor blockade. These findings indicate that Dyn release is tonically inhibited by PACAP and that blocking this inhibition, which increases the spinal release of Dyn, results in antinociception. Consistent with this conclusion, we found in the spinal dorsal horn that Dyn-immunoreactive neurons 1) expressed PAC(1)R and 2) were apposed by PACAP terminals. Present results, in combination with the previous demonstration that the release of spinal Dyn is tonically inhibited by opioid- and nociceptin/orphanin FQ-coupled pathways (J Pharmacol Exp Ther 298:1213-1220, 2001), indicate that spinal Dyn-ergic neurons integrate multiple inhibitory inputs, the interruption of any one of which (i.e., disinhibition) is sufficient to enhance spinal Dyn release and generate antinociception. Gaining a better understanding of the role of primary afferent neurotransmitters in negatively modulating the spinal release of Dyn and the physiological use of disinhibition to increase spinal Dyn activity could suggest novel clinically useful approaches for harnessing endogenous Dyn for pain control.

Supraspinal Flumazenil Inhibits the Antianalgesic Action of Spinal Dynorphin A [1–17

DynorphinA (Dyn) administered intrathecally or released spinally in mice produces antianalgesia, that is, antagonizes morphine analgesia (tail-flick test). Spinal transection eliminates this Dyn antianalgesia. Present results in mice show that intracerebroventricular administration of flumazenil, a benzodiazepine receptor antagonist, also eliminated the antianalgesic action of Dyn; flumazenil in the brain eliminated the suppressant effect of intrathecal Dyn on intrathecal and intracerebroventricular morphine-induced antinociception. Intracerebroventricular clonidine, naloxone, and norbinaltorphimine release spinal Dyn. The latent antinociceptive actions of these compounds were uncovered by intracerebroventricular flumazenil. Thus, Dyn, given intrathecally or released spinally, activates a pathway that is inhibited by intracerebroventricular flumazenil. Dyn antianalgesia is not significantly altered by intracerebroventricular administration of bicuculline and picrotoxin, suggesting that activation of the gamma-aminobutyric acid receptor has little if any involvement in the antianalgesic action of Dyn. The antagonistic effect of Dyn seems to be mimicked by benzodiazepine agonists. Furthermore, administration of a benzodiazepine receptor inverse agonist (methyl-6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate) inhibited Dyn antianalgesia as did flumazenil. Thus, flumazenil, through a benzodiazepine antagonist or inverse agonist action, interrupts, as does spinal transection, the neuronal circuit (cord/brain/cord) necessary for the antianalgesic action of spinal Dyn. Because Dyn antianalgesia is an indirect action, activation of the neuronal circuit must lead to the release of a direct-acting antianalgesic mediator in the spinal cord.

Dynorphin neurotoxicity induced nitric oxide synthase expression in ventral horn cells of rat spinal cord

Nitric oxide (NO) mediation in the spinal cord injury induced by intrathecal (i.t.) dynorphin (Dyn) administration was studied with NADPH-diaphorase (Nd) histochemistry. Normally, there was rarely NO synthase (NOS) activity in spinal cord motoneurons, and Dyn A(1–17) 10 nmol, which produced only transient paralysis, did not induce Nd/NOS expression in ventral horn cells. After a paralyzing dose of i.t. Dyn A(1–17) 20 nmol, which definitely produced permanent paraplegia and neuronal death, Nd/NOS began to express in motoneurons at 30 min, increased in numbers and intensities at 2–4 h and persisted up to 8 h. Most of Nd/NOS motoneurons disappeared at 24 h coincident with the neuronal death. Quite a few intensively-stained Nd-positive small cells and swollen varicosities became visible only in rats with permanent paraplegia and neuronal death, beginning at 2 h, maximizing at 3–4 h and remaining up to 24 h. These results suggest that NOS expression was induced in the ventral horn of spinal cord, including small cells and varicosities as well as motoneurons closely correlated in time and degree with pathological changes in motoneurons caused by spinal Dyn neurotoxicity.

Inhibiting a Spinal Dynorphin A Component Enhances Intrathecal Morphine Antinociception in Mice

Morphine given intracerebroventricularly releases spinal dynorphin A (Dyn) in mice. The present study was undertaken to determine whether morphine given intrathecally (IT) released Dyn. We demonstrated that the antinociceptive action of morphine was enhanced by procedures that are known to attenuate Dyn action. First, coadministration of the opiate antagonists, naloxone (5 fg), norbinaltorphimine (5 fg) or beta-funaltrexamine (0.25 ng) with IT morphine (0.15 microgram, 5 min) increased antinociceptive percentage maximum possible effect (%MPE) from 30% to 65%. Second, dynorphin antiserum (5 micrograms, 1 h, IT), which neutralizes Dyn action, enhanced morphine (0.2 microgram, 5 min, IT) action; MPE of 27% was increased to 60%. Third, production of desensitization to the antagonistic action of Dyn, IT, by pretreatment with morphine [10 mg/kg, 3 h, subcutaneously (SC)], or 2 micrograms, 3 h, IT) or Dyn (1 ng, 1 h, IT) increased the 30% MPE of IT morphine to 60%. Naloxone [1 ng/kg, intraperitoneally (IP)] enhanced IT morphine at a peak time of 20 min. Nalmefene [1 to 100 ng/kg, per os (PO)] enhanced IT morphine action. In conclusion, the present study showed that IT morphine putatively released spinal Dyn.

Inhibition of the antianalgesic action of dynorphin A in mice by cholera toxin.

Dynorphin A-(1-17) (Dyn A) administered intrathecally (IT) or released spinally in the mouse produces an antianalgesic action. The present experiments indicate that IT administration of cholera toxin inhibited the antianalgesic action of Dyn A. When clonidine was administered intracerebroventricularly (ICV) to release spinal Dyn A, IT cholera toxin inhibited the antianalgesic action of Dyn A so that the analgesic component of action of clonidine became evident. Dyn A given IT inhibited the analgesic action of morphine given ICV. Cholera toxin given IT eliminated the antagonistic action of Dyn A. These results, in addition to others, indicate that IT cholera toxin antagonized the action of Dyn A. When the antianalgesic action of Dyn A was attenuated by pretreatment with dynorphin antiserum or by pretreatment that produced desensitization to Dyn A, cholera toxin had no effect. These results suggested that the antianalgesic action of Dyn A is mediated by activation of opioid receptors that are positively coupled to adenylate cyclase through a Gs regulatory protein.

Dynorphin A(1–17) mediates midazolam antagonism of morphine antinociception in mice

Studies have shown that midazolam acts in the brain to antagonize the antinociception produced by morphine. The purpose of this study was to determine if spinal dynorphin A(1–17) (Dyn) was involved in the antagonistic effects of midazolam. A number of drugs when administered intracerebroventricularly (ICV) to mice release Dyn in the spinal cord to antagonize morphine-induced antinociception. In the present study using the mouse tail-flick test, midazolam administered ICV produced a dose related reduction of the antinociception induced by morphine given intrathecally (IT). The antagonistic action of midazolam against morphine-induced antinociception involved the release of Dyn in the spinal cord, as evidenced by the following results. 1) Administration of naloxone, norbinaltorphimine and dynorphin antiserum, IT, eliminated the antagonistic effect of midazolam, given ICV, against morphine. Treatment with these opioid antagonists and dynorphin antiserum is known to inhibit the action of spinally released Dyn. 2) Production of desensitization to the effect of spinal Dyn by pretreating with morphine, 10 mg/kg subcutaneously 3 h before the tail-flick test, abolished the antagonistic action of midazolam given ICV. A 3-h pretreatment with midazolam, ICV, also produced desensitization to the antianalgesic action of Dyn given IT. 3) Elimination of the Dyn component of action of midazolam by administration of naloxone, nor-binaltorphimine and dynorphin antiserum, IT, uncovered slight antinociceptive activity of midazolam, given ICV. Coadministration of flumazenil (a benzodiazepine antagonist), bicuculline (a GABA antagonist) and picrotoxin (a chloride ion channel blocker) inhibited the midazolam effect. Thus, activation of the supraspinal GABA/benzodiazepine receptor complex by midazolam appeared to be involved in the antagonistic action of midazolam and the release of spinal Dyn.

Opioid antagonists: indirect antagonism of morphine analgesia by spinal dynorphin A

Naloxone and norbinaltorphimine when given ICV ICV to mice can antagonize IT morphine-induced analgesia indirectly by releasing spinal dynorphin A(1–17) (Dyn A). Dyn A produces an antianalgesic action against IT morphine. In the present study, drugs with varying amounts of opioid antagonist to agonist action (nalbuphine, levallorphan, naltrexone, and naltrindole) were given ICV to determine whether they antagonized IT morphine-induced inhibition of the tail-flick response as an indication of spinal Dyn A release. Additional pharmacological tests were used as criteria for Dyn A release: a) Small doses of the opioid antagonists naloxone and norbinaltorphimine administered IT inhibited the antagonistic action; b) dynorphin antiserum given IT blocked the action of Dyn A; c) desensitization to the effect of Dyn A was produced by 3-h pretreatment with morphine, 10 mg/kg SC, or by pretreatment with the agents themselves. When given ICV, nalbuphine, levallorphan, and naltrexone released Dyn A in the spinal cord to produce an antianalgesic effect. Naltrindole, a δ-receptor antagonist, did not release Dyn A. Dyn A release did not appear to involve δ-receptors. Thus, a number of opioid antagonists inhibit the analgesic action of opioid agonists indirectly through Dyn A release.

Spinal cord dynorphin may modulate nociception via a ℵ-opioid receptor in chronic arthritic rats

Inoculation of rats with Mycobacterium butyricum produced an arthritis of the limbs which revealed an enhanced sensitivity to noxious mechanical pressure (hyperalgesia). Arthritic rats displayed a pronounced rise in immunoreactive dynorphin in lumbo-sacral spinal cord which correlated both with the intensity and time-course of this hyperalgesia. MR-2266, a relatively preferential antagonist at the ℵ-opioid receptor (at which dynorphin is considered to act) potentiated this hyperalgesia. In contrast, MR 2267 (its inactive stereoisomer) was ineffective. Further, naloxone (a weak ℵ-antagonist), and ICI 154,129 (a preferential σ-antagonist) were, in each case, inactive. The data demonstrate a pronounced response of spinal dynorphin to chronic arthritic pain in the rat. In addition, they raise the possibility of a function of spinal DYN, via a ℵ-receptor, in the modulation of chronic arthritic pain.