Thomas R. Tölle

The distribution of splice variants of the NMDAR1 subunit mRNA in adult rat brain.

Regional variation in the alternative splice forms of the NMDAR1 subunit mRNA was investigated by in situ hybridization in the adult rat brain, using radiolabelled splice-specific oligonucleotide probes. Each splice variant was detected in an individual distribution. The NMDAR1-a and NMDAR1-2 forms were widely and abundantly distributed throughout the brain, except for the inferior colliculus. The NMDAR1-b and NMDAR1-4 variants were located in similar patterns in fewer areas (e.g. parietal cortex, hippocampus CA3, thalamus, inferior colliculus, cerebellar granule cells). In contrast, the NMDAR1-1 forms were distributed in a pattern approximately complementary in the forebrain to that of NMDAR1-4 (weakly expressed in thalamus and inferior colliculus). The NMDAR1-3 variants were not abundant in any structure. Considerable overlap of the in situ hybridization images was noted, so all eight splice combinations are possible in heterogenous distributions. Correlation of the distribution of NMDAR1 mRNA splice forms with functional analyses of heteromeric recombinant receptors will be necessary to ascertain if alternative splicing of the NMDAR1 subunit can account for some of the known heterogeneity of endogenous NMDA receptors.

Opiates modify induction of c-fos proto-oncogene in the spinal cord of the rat following noxious stimulation

The expression of the proto-oncogene c-fos in neurons of the spinal cord dorsal horn of the rat following noxious thermal stimulation was compared in morphine- and ketamine-treated animals. Intravenous injection of morphine reduced the number of c-fos-positive neurons by up to 85% in laminae III-VI and X. This effect was dose dependent and naloxone reversible. The non-competitive N-methyl-D-aspartate (NMDA) antagonist ketamine had no effect. The present data show that morphine suppresses the induction of c-fos. A block of Ca2+ influx through voltage- and ligand (NMDA)-gated channels does not influence c-fos protein synthesis in the dorsal horn of the spinal cord in vivo.

Increase in GABAergic Cells and GABA Levels in the Spinal Cord in Unilateral Inflammation of the Hindlimb in the Rat

The effects of chronic peripheral inflammation on spinal cord γ‐aminobutyric acid (GABA) were examined in the rat. Following the injection of complete Freunds adjuvant in the left hindlimb footpad an increased number of immunoreactive cells occurred in ipsilateral laminae I‐III of the dorsal horn from L3 to L5. GABA‐immunoreactive cells were more numerous than contralaterally 1 week after the onset of the inflammation, reached maximal numbers after 3–4 weeks, and declined thereafter. Differences from control sides were statistically significant except at week 6. GABA levels in homogenates of the ipsilateral lumbar enlargement were increased significantly at 4 weeks. Since increases in GABA occurred in the spinal cord zone of projection of the nerves supplying the inflamed foot, the central response is surmised to result from the increased nociceptive input arriving from the periphery. However, the transmission from primary axons to GABA interneurons is not likely to be monosynaptic since profiles containing glutamate decarboxylase or GABA immunoreactivity are known to be predominantly presynaptic, and rarely postsynaptic, to primary afferent endings in electron micrographs in the rat. The findings support the function attributed to spinal GABA in modulating nociceptive input at segmental level.

Sequential expression of JUN B, JUN D and FOS B proteins in rat spinal neurons: Cascade of transcriptional operations during nociception

Expression of the immediate-early gene encoded proteins JUN B, JUN D and FOS B was investigated by immunocytochemistry in rat L5 spinal cord up to 24 h following stimulation of hind limb somatosensory nociceptors by noxious heat or injection of formalin. In both experimental protocols, JUN B, which did not show basal expression, reached maximum expression after 2 h and thereafter slowly decreased. In contrast, the expression of JUN D, which was present before stimulation in many spinal neurons, was increased after 4 h, reached its maximum after 8 h and thereafter remained elevated. FOS B which was absent under basal conditions reached its maximum between 4 h and 8 h and thereafter declined but was still present after 24 h. All immunoreactivities were restricted to the ipsilateral dorsal horn except JUN D which was also induced in the contralateral side after 8 h. The results are discussed in respect to their meaning for transcriptional operations of JUN and FOS proteins.

Enhanced expression of metabotropic glutamate receptor 3 messenger RNA in the rat spinal cord during ultraviolet irradiation induced peripheral inflammation

Metabotropic glutamate receptors are thought to play a role in the development and maintenance of spinal hyperexcitability resulting in hyperalgesia and pain. In this study we have used in situ hybridization to investigate the distribution of metabotropic glutamate receptors mGluR1-7 messenger RNA in the rat spinal cord in a model of inflammatory hyperalgesia. Hyperalgesia was induced in nine-day-old rats by exposure of the left hindpaw to an ultraviolet light source. Lumbar portions of spinal cords were removed from control and ultraviolet-treated animals. In situ hybridization with specific oligonucleotide probes was used to localize metabotropic glutamate receptor messenger RNAs. mGluR1, 3-5 and 7 subtype messenger RNA was detected in the gray matter of the spinal cord with distribution being specific for the different subtypes. A significant increase in the expression of mGluR3 messenger RNA was seen in cells of the dorsal laminae in both sides of the lumbar spinal cord. This increase was most pronounced in laminae II, III and IV but gradually decreased and disappeared by the third day of inflammation. In parallel with this, behavioural experiments revealed mechanical hyperalgesia in both hindlimbs after ultraviolet irradiation. There was no change in mGluR3 messenger RNA expression in the thoracic segments. No changes have been detected in the levels of expression of mGluR 1,2,4,5,7 subtype messenger RNA in spinal cords taken from hyperalgesic animals. These observations show that during ultraviolet irradiation induced inflammation, the synthesis of mGluR3 messenger RNA is altered suggesting that regulation of metabotropic glutamate receptor expression may be instrumental in plastic changes within the spinal cord during the development of hyperalgesia and pain.

Effects of Kelatorphan and morphine before and after noxious stimulation on immediate-early gene expression in rat spinal cord neurons.

&NA; Expression of the immediate‐early genes (IEG) c‐FOS, NGF1‐A and c‐JUN was induced hy noxious thermal stimulation in neurons of the rat spinal cord dorsal horn. Intravenous injection of Kelatorphan (5, 10 and 20 mg/kg), an inhibitor of multiple enkephalin‐degrading enzymes, 20 min before noxious stimulation reduced the overall number of dorsal horn neurons expressing c‐FOS and NGF1‐A by up to 20–30%. While c‐FOS expression was suppressed in superficial and deep laminae of the spinal cord, NGF1‐A and c‐JUN was only suppressed in superficial laminae. Morphine (5, 7.5 and 10 mg/kg) produced a dose‐dependent reduction of c‐FOS expression by up to 70% only when injected before noxious stimulation. Morphine injected K) min after the noxious treatment was virtually ineffective. The depressant effect of Kelatorphan and morphine could be prevented by prior application of the opioid antagonist naloxone. Naloxone itself slightly increased the overall number of c‐FOS‐positive neurons in all laminae of the spinal cord. The present data support the existence of a tonic release of endogenous opioid peptides at the spinal level and show that inhibition of their peptidase‐induced degradation modulates IEG expression in dorsal horn neurons of the rat. The finding that opioid agonists were ineffective when applied after stimulation underline the necessity of pre‐emptive analgesia to prevent long‐term activity‐dependent changes in spinal cord neurons.

Morphine, 5-HT2 and 5-HT3 receptor antagonists reduce c-fos expression in the trigeminal nuclear complex following noxious chemical stimulation of the rat nasal mucosa.

Noxious chemical stimulation of the rat nasal mucosa induces the expression of the immediate early gene c-fos in trigeminal brainstem neurons. In the present study, we applied the irritant mustard oil (1%) into the left nostril of urethane anesthetized rats. Immunohistochemical methods were used to evaluate the expression of Fos protein in the trigeminal subnuclei interpolaris and caudalis and to test the effects of putative analgesics that might depress synaptic transmission in neurons related to nociception. For this purpose, morphine (3 mg/kg and 10 mg/kg), the 5-HT2 antagonist ketanserin (0.5 mg/kg and 5 mg/kg) and the 5-HT3 antagonist ICS 205-930 (0.1 mg/kg and 1 mg/kg) were administered intravenously prior to noxious stimulation. Pretreatment with any of the three compounds reduced Fos-like immunoreactivity. The effect of morphine was reversible with naloxone. The reduction of the expression of Fos-like immunoreactivity by exogenous morphine speaks in favour of an opioidergic link in the modulation of orofacial pain in the trigeminal nuclei. The effects of the 5-HT receptor antagonists are most likely mediated via 5-HT2 and 5-HT3 receptors located on primary afferent fibres.

Carrageenan-induced inflammation of the hind foot provokes a rise of GABA-immunoreactive cells in the rat spinal cord that is prevented by peripheral neurectomy or neonatal capsaicin treatment

&NA; An increase in the number of &ggr;‐aminobutyric acid (GABA)‐immunorcactive cells is reported in the superficial dorsal horn of the rat spinal cord upon unilateral inflammation of the hind foot caused by subcutaneous carrageenan injection. The rise of GABAergic cells was restricted to the ipsilateral dorsal horn, reaching a peak value of 23.4% over the contralateral side 4 days after carrageenan injection. Sciatic neurectomy or neonatal capsaicin treatment prevented this effect. These findings suggest that dorsal horn GABA is up‐regulated by the increase of noxious inflow conveyed by unmyelinated C fibers from the inflamed tissues.

Application of morphine prior to noxious stimulation differentially modulates expression of FOS, JUN and KROX-24 proteins in rat spinal cord neurons

The expression of Fos, Jun and Krox-24 proteins was investigated in spinal cord neurons of the rat 2, 4 and 8 h following noxious thermal stimulation of one hind-paw and pre-treatment with morphine. The number of neurons expressing c-Fos, c-Jun, Jun B and Krox-24 were maximal after 2 h and thereafter declined. The number of Fos B and Jun D immunoreactive neurons increased constantly for up to 8 h with Jun D showing expression above baseline only after 4 h following stimulation. Intravenous application of morphine (5 and 10 mg/kg) 20 min before noxious heat stimulation decreased the expression of all six proteins at any time-point with a predilective effect on neurons of deeper laminae of the dorsal horn. The suppressive effects of morphine were more pronounced with the higher dose of morphine and completely reversed by intravenous naloxone (1 and 10 mg/kg). The temporospatial patterns of expression following morphine were similar to those seen without morphine, but in a much smaller number of neurons and with a shorter time-course. However, despite the high dose of morphine and continuous halothane anaesthesia during the whole experimental procedures, a considerable number of neurons expressing the various genes remained in all laminae of the spinal cord. At 2 h following noxious heat stimulation morphine had decreased the number of labelled neurons for c-Fos, Fos B, Krox-24, c-Jun and Jun B to 30-60% of control levels in laminae I-II and to 10-30% in laminae III-VII,X of the spinal cord. At 4 h the level of reduction had further increased while Jun D was only moderately reduced to 75% in all laminae of the spinal cord. Eight hours following noxious heat plus morphine application we did not detect noxious evoked immunoreactivity for c-Fos, Krox-24, c-Jun and Jun B, while there was residual labelling for Fos B in the superficial dorsal horn and for Jun D in laminae I-VII and X of the spinal cord. The different temporospatial pattern of immediate early gene expression in neurons of the spinal cord dorsal horn following noxious stimulation suggest that variable transcription complexes may interact with DNA regulatory sequences and could thus activate alternative secondary response genes, even under protection of a high dosage of morphine applied before noxious stimulation.

Expression of GAD mRNA in spinal cord neurons of normal and monoarthritic rats

This study was carried out to investigate whether the increase of GABA levels in spinal cord dorsal horn in response to chronic inflammatory lesions results from an enhanced expression of the gene that governs the production of glutamate decarboxylase (GAD), the enzyme responsible for GABA synthesis. In situ hybridization was used to visualize neurons expressing GAD mRNA within the spinal cord, in both intact rats and in animals bearing chronic monoarthritis induced by intraarticular injection of complete Freunds adjuvant. In control normal animals, neuronal labeling by an antisense oligonucleotide probe occurred throughout the spinal gray matter, except in the motoneuronal pool of Rexeds lamina IX. In treated animals 4 days after the induction of monoarthritis, a significant increase in the number of labeled cells occurred in the superficial laminae (25.3%) and the neck (17.2%) of the ipsilateral dorsal horn at segments L4-L5 which contain the projection domain of the ankle joint. At 2 weeks, values were, respectively, 20.2% and 13.9% over contralateral values, and an increase of 12.4% was found in the ventral horn. At 3 weeks, the ipsilateral increase of labeled cells was restricted to the superficial dorsal horn (15.2%). These findings emphasize the role played by the spinal GABAergic system in the modulation of chronic nociceptive input. It is suggested that the response of the spinal GABAergic system depends on the activation of GAD gene transcription in spinal neurons.