Regional distribution of acid phosphatase-positive axonal systems in the rat spinal cord and medulla,representing central terminals of cutaneous and visceral nociceptive neurons

Summary In addition to the substantia gelatinosa Rolandi, acid phosphatase active axonal systems are described (1) in the viscerosensory nucleus of the vagus nerve, (2) in Lissauer's band, (3) in the fasciculus cornus posterions (Cajal), and (4) in the nucleus basilaris externus (Cajal). Electron microscopically, acid phosphatase is located in between synaptic vesicles of axon terminals; the vesicle population of such terminals in the Rolando substance, however, markedly differs from that in systems 1–4, characterized by the presence of large dense-core vesicles. While acid phosphatase-active axon terminals in the Rolando substance appear to subserve cutaneous nociception, circumstantial evidence suggests participation of systems 1–4 in processing visceral nociception.     

Nociceptive spinal withdrawal reflexes but not spinal dorsal horn wide-dynamic range neuron activities are specifically inhibited by halothane anaesthesia in spinalized rats

The aim of the present study was to investigate the spinal cord effects and sites of action of different inhaled concentrations (0.5-2%) of the anaesthetic, halothane. Simultaneous recordings were made of 3 Hz, suprathreshold (1.5 x T) electrically evoked spinal dorsal horn (DH) wide-dynamic range (WDR) neuron responses and of single motor unit (SMU) electromyographic (EMG) responses underlying the spinal withdrawal reflex in spinalized Wistar rats. Compared with the baseline responses obtained with 0.5% halothane, the electrically evoked early responses of the DH WDR neurons as well as the SMUs were only depressed by the highest, 2% concentration of halothane. In contrast, 1.5% halothane markedly inhibited the late responses of the DH WDR neurons, whereas 1% halothane started to significantly depress the late responses of the SMUs. Likewise, wind-up of the WDR neuron late responses was inhibited by 1.5-2% halothane, whereas 1-2% halothane significantly depressed wind-up of the SMU EMG late responses. The inhibitory effects of 2% halothane on the early and the late responses of the DH WDR neurons, but not of the SMUs, were completely reversed by opioid micro-receptor antagonist naloxone (0.04 mg/kg). However, no significant effects of naloxone were found on different responses of the DH WDR neurons as well as the SMUs at 0.5-1% halothane, suggesting that different concentrations of halothane may modulate different spinal receptors. We conclude that halothane at high concentrations (1.5-2%) seems to play a predominant inhibitory role via spinal multireceptors on ventral horn (VH) motor neurons, and less on DH sensory WDR neurons, of the spinal cord.     

Prostaglandin-mediated control of rat brain kynurenic acid synthesis – opposite actions by COX-1 and COX-2 isoforms

Summary. Kynurenic acid (KYNA), an endogenous glutamate-receptor antagonist preferentially blocking NMDA-receptors, has analgesic properties and has also been implicated in the pathophysiology of schizophrenia. Recently, the non-steroid anti-inflammatory drug (NSAID) diclofenac was found to increase rat brain KYNA. Here, we analyze whether cyclooxygenase (COX)-1 or COX-2 modulate the levels of rat brain KYNA. The non-selective COX-inhibitor diclofenac (50mg/kg, i.p.) or indomethacin (50mg/kg, i.p.), a non-selective inhibitor with a preferential selectivity for COX-1, produced an elevation in brain KYNA. In contrast, the COX-2 selective inhibitors parecoxib (25mg/kg, i.p.) or meloxicam (5mg/kg, i.p.) decreased brain KYNA. Both elevation and lowering of brain KYNA by indomethacin or parecoxib, respectively, were prevented by the prostaglandin E₁/E₂ agonist misoprostol (1mg/kg, s.c.). It is proposed that increased brain KYNA formation induced by NSAIDs displaying an inhibitory action on COX-1 contribute to their analgesic efficacy as well as to their psychiatric side effects.     

Characteristics of the trigeminal depressor response in cats

We studied the effects of electrical stimulation of the inferior alveolar nerve (IAN) on cardiovascular responses in cats. There was statistical correlation between cardiovascular response and prestimulus mean arterial blood pressure (MABP) and heart rate (HR). A trigeminal depressor response (TDR) was induced when the prestimulus MABP and HR were above 95 mm Hg and 140 beats/min, respectively. We investigated further to identify the vasomotor regulating center and neural transmitters involved in TDR. In the medulla, electrical stimulation of the dorsomedial medulla, the infratrigeminal nucleus (IFT), and the rostral ventrolateral medulla (RVLM) induced a vasopressor response. We confirmed that neurons in the RVLM were retrogradely labeled by wheat germ agglutinin-conjugated horseradish peroxidase injection into the nucleus intermediolateralis of the spinal cord. The vasopressor response induced by IFT stimulation was similar to that induced by IAN stimulation. Vasodepressor responses were induced when the caudal ventrolateral medulla, the nucleus tractus solitarius, the lateral tegmental field, the trigeminal nucleus interpolaris, the trigeminal spinal tract, and the paramedian reticular nucleus were stimulated. These responses, however, were not similar to the vasodepressor response induced by IAN stimulation but were similar to the cardiovascular response induced by vagal afferent stimulation. After spinalization or lesion of the RVLM, MABP and HR decreased and TDR completely disappeared. Inhibitory synaptic ligands and receptors were localized using immunohistochemical techniques. Neurons immunopositive for adrenaline, noradrenaline, and gamma-aminobutyric acid (GABA), and adrenaline alpha(2A), GABA(A), GABA(B), and glycine receptors were distributed along the sympatho-reflexive route including the RVLM and IFT. These results suggest that TDR could be induced as negative feedback to sympathetic hyperactivity whenever MABP and HR are high, because of the inhibitory control of the RVLM.     

Bidirectional modulation of windup by NMDA receptors in the rat spinal trigeminal nucleus

Activation of afferent nociceptive pathways is subject to activity-dependent plasticity, which may manifest as windup, a progressive increase in the response of dorsal horn nociceptive neurons to repeated stimuli. At the cellular level, N-methyl-d-aspartate (NMDA) receptor activation by glutamate released from nociceptive C-afferent terminals is currently thought to generate windup. Most of the wide dynamic range nociceptive neurons that display windup, however, do not receive direct C-fibre input. It is thus unknown where the NMDA mechanisms for windup operate. Here, using the Sprague-Dawley rat trigeminal system as a model, we anatomically identify a subpopulation of interneurons that relay nociceptive information from the superficial dorsal horn where C-fibres terminate, to downstream wide dynamic range nociceptive neurons. Using in vivo electrophysiological recordings, we show that at the end of this pathway, windup was reduced (24 +/- 6%, n = 7) by the NMDA receptor antagonist AP-5 (2.0 fmol) and enhanced (62 +/- 19%, n = 12) by NMDA (1 nmol). In contrast, microinjections of AP-5 (1.0 fmol) within the superficial laminae increased windup (83 +/- 44%, n = 9), whereas NMDA dose dependently decreased windup (n = 19).These results indicate that NMDA receptor function at the segmental level depends on their precise location in nociceptive neural networks. While some NMDA receptors actually amplify pain information, the new evidence for NMDA dependent inhibition of windup we show here indicates that, simultaneously, others act in the opposite direction. Working together, the two mechanisms may provide a fine tuning of gain in pain.     

A comparative reappraisal of projections from the superficial laminae of the dorsal horn in the rat: the forebrain

Projections to the forebrain from lamina I of spinal and trigeminal dorsal horn were labeled anterogradely with Phaseolus vulgaris-leucoagglutinin (PHA-L) and/or tetramethylrhodamine-dextran (RHO-D) injected microiontophoretically. Injections restricted to superficial laminae (I/II) of dorsal horn were used primarily. For comparison, injections were also made in deep cervical laminae. Spinal and trigeminal lamina I neurons project extensively to restricted portions of the ventral posterolateral and posteromedial (VPL/VPM), and the posterior group (Po) thalamic nuclei. Lamina I also projects to the triangular posterior (PoT) and the ventral posterior parvicellular (VPPC) thalamic nuclei but only very slightly to the extrathalamic forebrain. Furthermore, the lateral spinal (LS) nucleus, and to a lesser extent lamina I, project to the mediodorsal thalamic nucleus. In contrast to lamina I, deep spinal laminae project primarily to the central lateral thalamic nucleus (CL) and only weakly to the remaining thalamus, except for a medium projection to the PoT. Furthermore, the deep laminae project substantially to the globus pallidus and the substantia innominata and more weakly to the amygdala and the hypothalamus. Double-labeling experiments reveal that spinal and trigeminal lamina I project densely to distinct and restricted portions of VPL/VPM, Po, and VPPC thalamic nuclei, whereas projections to the PoT appeared to be convergent. In conclusion, these experiments indicate very different patterns of projection for lamina I versus deep laminae (III-X). Lamina I projects strongly onto relay thalamic nuclei and thus would have a primary role in sensory discriminative aspects of pain. The deep laminae project densely to the CL and more diffusely to other forebrain targets, suggesting roles in motor and alertness components of pain.     

Different tumors in bone each give rise to a distinct pattern of skeletal destruction,bone cancer-related pain behaviors and neurochemical changes in the central nervous system

Pain is the most common presenting symptom in patients with bone cancer and bone cancer pain can be both debilitating and difficult to control fully. To begin to understand the mechanisms involved in the generation and maintenance of bone cancer pain, we implanted 3 well-described murine tumor cell lines, 2472 sarcoma, B16 melanoma and C26 colon adenocarcinoma into the femur of immunocompromised C3H-SCID mice. Although each of the tumor cell lines proliferated and completely filled the intramedullary space of the femur within 3 weeks, the location and extent of bone destruction, the type and severity of the pain behaviors and the neurochemical reorganization of the spinal cord was unique to each tumor cell line injected. These data suggest that bone cancer pain is not caused by a single factor such as increased pressure induced by intramedullary tumor growth, but rather that multiple factors are involved in generating and maintaining bone cancer pain.     

Intrathecally applied flurbiprofen produces an endocannabinoid-dependent antinociception in the rat formalin test

It is generally accepted that the phospholipase-A2-cyclooxygenase-prostanoids-cascade mediates spinal sensitization and hyperalgesia. However, some observations are not in line with this hypothesis. The aim of the present work was to investigate whether different components of this cascade exhibit nociceptive or antinociceptive effects in the rat formalin test. Intrathecal (i.th.) injection of prostaglandin E2 (PGE2) induced a dose-dependent antinociceptive effect on the formalin-induced nociception. Furthermore, thimerosal, which inhibits the reacylation of arachidonic acid thereby enhancing arachidonic acid levels, had an antinociceptive effect rather than the expected pronociceptive effect when given i.th. While the phospholipase A2 inhibitor methyl arachidonyl fluorophosphonate (MAFP; i.th.) had a significant antinociceptive effect, its analogue palmitoyl trifluoromethyl ketone (PTFMK; i.th.) had no significant effect on the formalin-induced nociception. However, MAFP, but not PTFMK, showed a cannabinoid CB1 agonistic effect as shown by the inhibition of electrically evoked contractions of the vas deferens isolated from CB1 wild-type mice but not of that from CB1 knockout mice. The antinociceptive effect of MAFP was completely reversed by the CB1 receptor antagonist AM-251 (i.th.), thus attributing such effect to its CB1 agonistic effect. Moreover, the antinociceptive effect of the cyclooxygenase inhibitor, flurbiprofen (i.th.) was reversed by the co-administration of AM-251, but not by PGE2. Finally. the combination of phenylmethylsulfonyl fluoride (PMSF; intraperitoneal), which inhibits the degradation of anandamide through the inhibition of fatty acid amidohydrolase, with thimerosal (i.th.) produced a profound CB1-dependent antinociception. The present results show that endocannabinoids play a major role in mediating flurbiprofen-induced antinociception at the spinal level.     

Acute antinociceptive responses in single and combinatorial opioid receptor knockout mice: distinct mu,delta and kappa tones

We have examined responses of mice lacking mu, delta and kappa opioid receptor (MOR, DOR and KOR, respectively) genes, as well as combinatorial mutants, in several pain models. This is the first truly comparative study of all three opioid receptor-deficient mice, with genotypes and gender analysis using mice on the hybrid 50% 129/SV : 50% C57BL/6 genetic background. In the tail-immersion test, only KOR-/- females showed decreased withdrawal latencies. This modification was also found in MOR/KOR and MOR/DOR/KOR, but not MOR/DOR mutants. The hotplate test revealed increased nociceptive sensitivity for MOR-/-, a phenotype which was also observed in double mutants involving the MOR deletion, and in the triple mutants. The tail-pressure test showed increased response for both MOR-/- and DOR-/- mutants, a modification which was enhanced in the triple-mutant mice. In the formalin test, MOR-/- and DOR-/- mice showed increased responses in the early and late phases, respectively, while the triple mutant tended to show enhanced nociception in both phases. Finally, the enhanced response of KOR-/- mice in the writhing test, which we have demonstrated previously, was confirmed in double MOR/KOR- and triple-mutant mice. Together, the data support the existence of an antinociceptive opioid tone. Each receptor presents a distinct pattern of activities, with mu receptors influencing responses to mechanical, chemical and thermal nociception at a supraspinal level, kappa receptors involved in spinally mediated thermal nociception and chemical visceral pain, and delta receptors modulating mechanical nociception and inflammatory pain. Phenotypes of mutant mice were subtle, suggesting a low endogenous opioid tone in the regulation of physiological pain.