Involvement of M3 muscarinic receptors of the spinal cord in formalin-induced nociception in mice

Subcutaneous injection of formalin into a paw of mice caused two distinct phases of licking and biting, first phase (1–5 min) and the second phase (7–30 min) after the injection. The muscarinic antagonist atropine (0.1–10 ng, i.t.) and the M₃ receptor antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) (0.1–20 ng, i.t.) inhibited the second phase of this response, whereas higher doses of atropine (20–100 ng, i.t.) did not cause inhibition. The M₁ muscarinic receptor antagonist pirenzepine (10–100 ng, i.t.) did not inhibit either the first or the second phase response, but a high dose of pirenzepine (1000 ng, i.t.) tended to inhibit the second phase response. On the other hand, the M₂ muscarinic receptor antagonist 11-{(2-[(diethylamino)methyl]-1-piperidinyl}acetyl)-5,11-dihydro-6H-pyrido(2,3-b)(1,4)benzodiazepine-6-one (AF-DX116; 10–1000 ng, i.t.) had no effect on either the first or the second phase of response. The opioid receptor antagonist naloxone did not affect the 4-DAMP-induced anti-nociceptive response. The i.t. injection of the acetylcholinesterase inhibitor neostigmine (25 ng) significantly inhibited only the second phase. The acetylcholine (ACh) depletor hemicholinium-3 (HC-3) (1 μg, i.t.) completely abolished the 4-DAMP-induced anti-nociceptive response. The ACh content of the spinal cord was significantly increased 14 min after formalin injection. This significant increase in the ACh content was inhibited by pretreatment with 4-DAMP (10 ng, i.t.). These results suggest that endogenous ACh in the spinal cord acts as a transmitter anti-nociception, and that ACh release regulated by presynaptic M₃ muscarinic receptors in the spinal cord is involved in the second phase of nociception induced by formalin.     

Regional distribution of cholinergic muscarinic receptors in spinal cord

Quinuclidinylbenzilate ([³H]QNB) binding sites are present in the rat spinal cord. The binding sites are muscarinic in character based on displacement of [³H]QNB by cholinoceptive drugs. They are distributed rather uniformly along the cord, although the receptor density is greater in gray matters than in white matter. Binding to white matter may be associated with glial cells. Within the gray matter, the receptor density is higher in the ventral born than in the dorsal horn. In the thoracic region receptor density is about equal in the intermediate zone and ventral horn. Midthoracic transection of the cord does not change the receptor density or the dissociation constant of [³H]QNB in the lumbar cord. In contrast, treatment with the neurotoxin, 6-aminonicotinamide, which produces lesions of the cord, loss of motor control and paralysis, reduces the receptor density and affinity of [³H]QNB for lumbar gray matter but not white matter. The presence of [³H]QNB binding sites throughout the spinal cord as well as the documented presence of acetylcholine-containing neurons, suggest that muscarinic receptors play a role in all phases of spinal cord physiology.     

Paradoxical sleep in mice lacking M3 and M2/M4 muscarinic receptors

Acetylcholine is crucial for the regulation of paradoxical sleep (PS) and EEG theta activity. To determine the contribution of individual muscarinic receptors to these events, we analyzed the sleep-waking cycle and EEG activities of mice lacking functional M(3) or M(2)/M(4 )receptors. Daily PS amounts were significantly decreased in M3-/- (-22%) but not in M2/M4-/- mice. Further, the theta peak frequency for PS was significantly increased in both M2/M4-/- and M3-/- mice. This study supports the potential role of M(3) rather than M(2) and M(4) muscarinic receptors in the modulation of PS in mice and strengthens the idea that multiple muscarinic receptors contribute to the regulation of the EEG theta activity during PS.     

Involvement of κ‐opioid receptors in visceral nociception in mice

Abstract It has been shown that the behavioural responses to chemically evoked visceral nociception are increased in transgenic mice lacking the κ‐opioid receptor (KOR). The aim of the present study was to evaluate the contribution of KOR in mechanically evoked visceral pain by performing colorectal distension (CRD) and monitoring the subsequent visceromotor response (VMR) in control mice (KOR^+/+) and in mice lacking KOR (KOR^?/?). Pseudo‐affective visceral pain responses were evoked in conscious mice using increasing (10–80 mmHg) and repeated (12 × 55 mmHg) phasic CRD paradigms. The resulting VMR was determined by monitoring the electromyographic activity of the abdominal muscle. The increasing and repeated CRD paradigms, respectively, evoked similar responses in both KOR^+/+ and KOR^?/? mice. The selective KOR‐agonists U‐69593 (5 and 25 mg kg^?1, s.c.) and asimadoline (25 mg kg^?1, s.c.) significantly decreased the VMR in KOR^+/+ mice, while having no effect in KOR^?/? mice. In contrast, the selective μ‐opioid receptor agonist fentanyl significantly reduced the VMR in both types of mice and appeared more efficacious in KOR^?/? mice. The opioid receptor antagonist naloxone (0.3–30 mg kg^?1 s.c.) did not affect the response to CRD in C57BL/6 mice at any dose tested. In conclusion, the data confirm that the KOR agonists used in this study inhibit the VMR to CRD in mice by acting via KOR receptors. In addition, the data suggest that the endogenous opioid system is not likely to modulate the VMR to mechanically evoked visceral pain in mice.     

Loss of muscarinic M4 receptors in spinal cord of arthritic rats: implications for a role of M4 receptors in pain response

Changes in the levels of muscarinic M4 receptors in spinal cord of acute and chronic arthritic rats (animal models of pain) were studied by receptor autoradiography using muscarinic M4 receptor subtype selective ligand. Arthritis was induced in female Lewis rats by single intradermal injection of heat-killed Mycobacterium butyricum and sacrificed 12 days (acute group) and 30 days (chronic and control groups) after induction of arthritis. Our results demonstrate significant reduction in the level of M4 receptors in the spinal cord (Rexed laminae I-X) of acute and chronic arthritic rats compared to controls. These findings suggest that the muscarinic M4 receptor subtype may be involved in cholinergic mechanisms of analgesia.     

Toluene exposure enhances acute and chronic formalin-induced nociception in rats: Participation of 5-HT3 receptors

The purpose of this study was to evaluate the effect of acute toluene exposure on formalin (0.5% and 1%)-induced acute and long-lasting nociceptive hypersensitivity in rats. In addition, we sought to investigate the role of peripheral 5-HT₃ receptors in the pronociceptive effect of toluene. Toluene exposure (6000 ppm) for 30 min enhanced 0.5% or 1% formalin-induced acute nociception and long-lasting secondary allodynia and hyperalgesia. In contrast, exposition to toluene for 30 min in rats previously injected (six days before) with 1% formalin did not affect long-lasting hypersensitivy. Local peripheral pre-treatment with alosetron (5-HT₃ receptor antagonist, 10–100 nmol) reduced the pronociceptive effect of toluene in acute nociception and long-lasting secondary allodynia and hyperalgesia. Alosetron (100 nmol) was also able to reduce the nociceptive effects of 1% formalin in absence of toluene. Moreover, local peripheral injection of m-CPBG (5-HT₃ receptor agonist, 300 nmol) enhanced 0.5% formalin-induced acute and long-lasting nociception in air- and toluene-exposed rats. Alosetron (10 nmol) blocked the pronociceptive effects of m-CPBG (300 nmol) on 0.5% formalin-induced acute and long-lasting hypersensitivity in rats exposed to toluene. Alosetron (at 10 nmol) did not modify formalin-induced nociceptive behaviors. Finally, local peripheral pre-treatment with methiothepin (non-selective 5-HT receptor antagonist, 1.5 nmol), did not affect the pronociceptive effect of toluene on 1% formalin-induced acute and long-lasting hypersensitivity. Our data demonstrate that acute exposure to toluene has pronociceptive effects in formalin-induced acute nociception and long-lasting hypersensitivity. Our data suggest that this pronociceptive effect depend on activation of peripheral 5-HT₃, but not methiothepin-sensitive 5-HT, receptors.     

Pain, nociception and spinal opioid receptors

1. Opioid peptides derived from proenkephalin and prodynorphin are differentially distributed in the spinal cord. Proenkephalin peptides are preferentially located in the sacral portion of the cord while prodynorphin peptides are concentrated in the cervical spinal cord.

2. μ opioid receptor are highly concentrated in superficial layers of the dorsal horn in all the spinal cord.

3. δ opioid receptor are more diffusely distributed in the gray matter of the spinal cord. These sites are principally located in cervical and thoracic portions of the spinal cord.

4. κ opioid receptors are highly concentrated in the superficial layers of the lumbo-sacral spinal cord. Its density decreased in the upper levels of the spinal cord.

5. It appears that μ opioid receptors are indifferentially activated by thermal, pressure and visceral nociceptive inputs. δ receptors are more likely to be involved in thermal nociception while κ opioid binding sites are associated to visceral pain nociceptive inputs.     

Involvement of the spinal cord in Parkinson's disease

Parkinson's disease (PD) has traditionally ascribed to alpha-synucleinopathy of the substantia nigra; however, several studies have showed widespread alpha-synucleinopathy inside and outside the brain. The potential role of the spinal cord in the genesis of some symptoms has been quite neglected despite the frequent and precocious presence of alpha-synucleinopathy in the spinal cord of PD patients. We examined the literature about the pathology of the spinal cord in PD and the possible relevance of this pathology in the genesis of some nonmotor symptoms including urinary, sexual, and gastrointestinal, as well as of some motor symptoms.     

Effects of intrathecal amylin on formalin-induced nociception and on cAMP accumulation in the rat embryonic spinal cells

Amylin (AMY) is a member of calcitonin family of peptides. In this study, the effects of intrathecal (i.t) injection of AMY on the inflammatory pain and on the cAMP accumulation in the rat spinal cells were investigated. By using AMY receptor antagonists, we also studied the pharmacology of AMY receptors in the spinal cells. Formalin model of inflammatory pain was induced by intraplantar injection of formalin. AMY (0.06250–2500 pmol/rat) was administrated i.t 15 min before the injection of formalin. Antagonists were injected i.t 10 min before the injection of AMY and/or morphine. AMY reduced formalin-induced pain in a dose dependent mode. This effect was inhibited by the potent AMY antagonist, AC187 but not CGRP_8-37. rAMY_8-37, most commonly reported as a weak AMY antagonist, showed to be equally or more potent than AC187 in antagonizing the above effects. The opioid antagonist, naloxone, had no significant effects on AMY antinociceptive effects. Primary dissociated cell culture was used to investigate the effect of AMY on cAMP production and to characterize AMY receptors in the spinal cells. AMY moderately increases cAMP accumulation in the spinal cells with an EC₅₀ value of 74.62 nM. This effect was not affected by CGRP_8-37 but was inhibited by AC187 and rAMY_8-37 with pA₂ values of 7.94 and 7.87 respectively.In conclusion, effects of AMY in reducing formalin induced pain and on the cAMP accumulation by spinal cells are mediated through undefined receptors.     

Role of micro-opioid receptors in formalin-induced pain behavior in mice

Intraplantar formalin injection is widely used as an experimental model of tonic pain. We investigated the role of endogenous micro-opioid receptor mechanisms in formalin-induced nocifensive behavior in mice. The flinching response induced by formalin (2%, 20 microl) was studied in mice with normal (wild type, n = 8) and absent (homozygous micro-opioid receptor knockout, n = 8) micro-opioid receptor levels. The flinch responses were counted every 5 min for 60 min post-formalin injection. Lumbar spinal cord (L4, 5) was harvested 2 h post-formalin injection to examine c-Fos expression using immunohistochemistry. The effects of naloxone (5 mg/kg, sc) administered 30 min before the intraplantar formalin injection on the flinching response of wild-type mice (n = 7) were also recorded. The second-phase formalin response (10-60 min after formalin) was higher in homozygous micro-opioid receptor knockout mice compared to the wild-type mice (P < 0.01). Naloxone administration in wild-type mice before formalin injection resulted in pain behavior similar to that observed in homozygous micro-opioid receptor knockout mice (P > 0.05). The c-Fos expression induced by formalin injection in the knockout mice was not different from that observed in wild-type mice. Our results suggest that the endogenous micro-opioid system is activated by intraplantar formalin injection and exerts a tonic inhibitory effect on the pain behavior. These results suggest an important modulatory role of endogenous micro-opioid receptor mechanisms in tonic pain states.     

Gabapentin reverses mechanical allodynia induced by sciatic nerve ischemia and formalin-induced nociception in mice

The anticonvulsant drug gabapentin has been demonstrated to alleviate symptoms of painful diabetic neuropathy as well as other types of neuropathic pain. The aim of the present study was to investigate the effect of gabapentin in a recently developed mouse model of peripheral neuropathy. This model is based on a photochemical ischemic lesion of the sciatic nerve generated by laser-induced activation of the photosensitizing dye erythrosin B. Following laser irradiation of the sciatic nerve for 2, 5, or 10 min, tactile allodynia was observed during at least 3 weeks. The degree of allodynia was most marked following 10 min of irradiation. Subcutaneous administration of gabapentin [175-300 micromol/kg ( approximately 30-51 mg/kg), cumulative doses, at 1-h intervals] significantly reversed tactile allodynia induced by 10-min laser irradiation. The maximal dose of gabapentin increased the withdrawal threshold from approximately 0.55 to approximately 1.85 g (i.e., about 77% of the threshold in normal animals, approximately 2.4 g). Gabapentin did not affect the tactile withdrawal threshold in intact animals. A dose of gabapentin (100 micromol/kg, sc) that had no effect on allodynia was found to significantly reduce the pain behavior during phase 2 of the formalin test. The present study demonstrates that systemic administration of gabapentin suppresses both allodynia induced by an ischemic lesion of the sciatic nerve and pain behavior in the formalin test.     

Involvement of M1 and M2 muscarinic receptors of the basal forebrain in cholinergically mediated changes in the rat locomotion.

1. Wistar rats were implanted with cannulae into the medial preoptic and anterior hypothalamic areas for intracerebral injections. 2. Unilateral intracerebral injections of carbachol (1 microgram) into a limited area of the basal forebrain decreased significantly both the distance travelled by the animals and time spent walking. 3. In addition to the decrease in locomotion, it was demonstrated for the first time that injections of carbachol also slow animal movement by significantly decreasing speed of forward progression. 4. All locomotor effects of carbachol were reversed by local pretreatment with atropine (1 microgram) but not by pirenzepine (2 micrograms) suggesting that these effects were mediated by M2 muscarinic receptors with minimal or no M1 receptor involvement.     

M1 and M3 muscarinic receptors: specific roles in sleep regulation.

P-fluoro-hexahydro-sila-difenidol hydrochloride (p-F-HHSiD) (15, 30 micrograms) and pirenzepine (7.5, 15, 30 micrograms), which are highly selective M3 and M1 muscarinic antagonists, respectively, were injected intracerebroventricularly into freely moving rats. p-F-HHSiD (30 micrograms) reduced wakefulness (W) (from 34.7 +/- 3.1 to 24.9 +/- 1.3 min) and increased slow wave sleep (SWS) (from 56.7 +/- 2.4 to 67.2 +/- 1.5 min); however, it did not modify desynchronized sleep (DS) latency and percentage in 6 h recordings. W and SWS were not affected by pirenzepine (7.5, 15, 30 micrograms) which decreased significantly DS amount but left unaffected DS latency. The results suggest that each muscarinic receptor subtype may induce different and specific changes in sleep phases and cortical desynchronization processes.     

Experimental spinal cord injury: effects of trauma or ischemia on TRH and muscarinic receptors

Traumatic spinal cord injury in rats and ischemic spinal cord injury in rabbits were associated with time-dependent, localized decreases in thyrotropin-releasing hormone (TRH) and muscarinic receptor binding. Changes were not evident in the first 24 to 48 hours, consistent with the hypothesis that secondary alterations in spinal cord may occur relatively late after injury. TRH receptor binding, but not muscarinic receptor binding, recovered by 3 weeks following trauma. Since TRH and acetylcholine may serve as excitatory neurotransmitters within the spinal cord, such changes could contribute to the functional neurologic impairment that follows injury.     

Altered hippocampal muscarinic receptors in acetylcholinesterase-deficient mice

A primary therapeutic strategy for Alzheimer's disease includes acetylcholinesterase (AChE) inhibitors with the goal of enhancing cholinergic transmission. Stimulation of muscarinic acetylcholine receptors (mAChRs) by elevated levels of ACh plays a role in the effects of AChE inhibitors on cognition and behavior. However, AChE inhibitors only demonstrate modest symptomatic improvements. Chronic treatment with these drugs may cause mAChR downregulation and consequently limit the treatment efficacy. AChE knockout (-/-) mice were utilized in this study as a model for investigating the effects of selective, complete, and chronic diminished AChE activity on mAChR expression and function. In AChE -/- mice, the M(1), M(2), and M(4) mAChRs showed strikingly 50 to 80% decreased expression in brain regions associated with memory. In addition, mAChRs showed decreased presynaptic, cell surface, and dendritic distributions and increased localization to intracellular puncta. Furthermore, mAChR agonist-induced activation of extracellular signal-regulated kinase, a signaling pathway associated with synaptic plasticity and amyloidogenesis, is diminished in the hippocampus and cortex of AChE -/- mice. Therefore, chronic diminished ACh metabolism produces profound effects on mAChR expression and function. The alterations of mAChRs in AChE -/- mice suggest that mAChR downregulation may contribute to the limited efficacy of AChE inhibitors in Alzheimer's disease treatment.     

Involvement of 5-lipoxygenase in spinal cord injury

A traumatic spinal cord injury (SCI) induces a sequelae of events which conduce biochemical and cellular alterations. Here we compare the degree of spinal cord injury caused by the application of vascular clips, in mice lacking the 5-lipoxygenase and in the corresponding wild-type mice. Biochemical, immunohistochemical and functional studies revealed respectively an increase of neutrophils infiltration, of IL-1beta, TNF-alpha immunoreactivity, apoptosis (measured by Annexin-V staining) and loss of hind legs movement in SCI operated 5-LO wild-type mice. In contrast, the degree of (1) neutrophil infiltration at different time points, (2) cytokine expression (TNF-alpha and IL-1beta), (3) histological damage, (4) apoptosis, was markedly reduced in the tissues obtained from SCI operated 5-LO deficient mice and (5) the motor recovery was ameliorated.     

福尔马林致炎大鼠鞘内注射布托啡诺后脊髓背角NMDA受体的表达

研究背景与目的: 酒石酸布托啡诺是一种阿片类镇痛剂,对阿片受体具有激动/拮抗双重作用。其在临床和动物实验中的应用已有报道,但还未见鞘内应用于福尔吗林炎性痛大鼠模型的报道.另外在疼痛机制形成过程中,NMDA受体激活起着非常重要的作用。故我们假设鞘内注射酒石酸布托啡诺应用于福尔马林炎性痛大鼠时能产生明显的镇痛效果,其产生镇痛的机制可能是通过抑制NMDA受体表达来实现. 方法: 在大鼠的左后足掌面皮下注射5%福尔马林50μl致痛前30min,健康雄性SD大鼠接受生理盐水,低剂量布托啡诺（12.5μg）,高剂量布托啡诺（25μg）鞘内注射;对照组在福尔马林注射前不注射任何试剂,为了更进一步的研究,我们在设计中加入鞘内注射非选择性NMDA受体拮抗剂氯胺酮,也分别在福尔马林致痛前30min,鞘内注射低剂量（50μg）,高剂量（100μg）或低剂量（50μg）混合低剂量布托啡诺进行注射。与疼痛相关的行为学测定通过计算大鼠后爪理毛行为（后爪的离地、舔足/咬足）总计反应时间来确定,记录福尔马林诱导出注射后爪的双相理毛行为时间(在福尔马林注射后第1时相, 0-5 min; 第 2时相, 10-60 min ).所有大鼠均在注射后2h处死,用免疫组化法测定大鼠L5节段脊髓背角NMDA受体的表达。 结果：鞘内单独用布托啡诺当剂量增加到25μg时,以及鞘内低剂量布托啡诺和氯胺酮联合使用时都引起第1和2时相的后爪理毛行为的总计反应时间明显减少;其余的组别对福尔马林1和2时相都没有明显影响。鞘内单独用高剂量布托啡诺以及低剂量布托啡诺和氯胺酮联合处理都可以显著降低福尔马林致痛大鼠L5脊髓背角NMDA受体表达。 结论：本研究结果揭示鞘内用布托啡诺能够对福尔马林诱导的疼痛产生明显的镇痛作用,并具有剂量依赖性,其镇痛机制可能是通过抑制NMDA受体激活产生;不过具体NMDA受体抑制机制是否与鞘内布托啡诺激活к受体或μ受体有关还需要更进一步的研究来确定。     

Postsynaptic M1 and M3 receptors are responsible for the muscarinic enhancement of retrograde endocannabinoid signalling in the hippocampus

The cholinergic system is crucial for higher brain functions including learning and memory. These functions are mediated primarily by muscarinic acetylcholine receptors (mAChRs) that consist of five subtypes (M(1)-M(5)). A recent study suggested a novel role of acetylcholine as a potent enhancer of endocannabinoid signalling that acts retrogradely from postsynaptic to presynaptic neurons. In the present study, we further investigated the mechanisms of this cholinergic effect on endocannabinoid signalling. We made paired whole-cell recordings from cultured hippocampal neurons, and monitored inhibitory postsynaptic currents (IPSCs). The postsynaptic depolarization induced a transient suppression of IPSCs (DSI), a phenomenon known to involve retrograde signalling by endocannabinoids. The cholinergic agonist carbachol (CCh) markedly enhanced DSI at 0.01-0.3 microM without changing the presynaptic cannabinoid sensitivity. The facilitating effect of CCh on DSI was mimicked by the muscarinic agonist oxotremorine-M, whereas it was eliminated by the muscarinic antagonist atropine. It was also blocked by a non-hydrolizable analogue of GDP (GDP-beta-S) that was applied intracellularly to postsynaptic neurons. The muscarinic enhancement of DSI persisted to a substantial degree in the neurons prepared from M1-knockout and M3-knockout mice, but was virtually eliminated in the neurons from M1/M3-compound-knockout mice. CCh still enhanced DSI significantly under the blockade of postsynatpic K(+) conductance, and did not significantly influence the depolarization-induced Ca(2+) transients. These results indicate that the activation of postsynaptic M1 and M3 receptors facilitates the depolarization-induced release of endocannabinoids.