Supersensitivity to the antinociceptive effect of a 5-HT1 receptor agonist after lesion of raphe-spinal serotonergic neurones.

The actions of the 5-hydroxytryptamine type-1B (5-HT1) receptor agonist 1(m-chlorophenyl)piperazine (mCPP) on response latencies in the tail-flick test and tail skin temperature were examined in mice after pretreatment with the 5-HT neurotoxin, 5,6-dihydroxytryptamine (5,6-DHT). All the compounds were given intrathecally. The level of 5-HT in spinal cord was reduced by 79% 4 days after intrathecal injection of 5,6-DHT (10 micrograms). There was a significant relationship between the tail-flick latency and tail skin temperature, and the reduced baseline tail-flick latencies induced by 5,6-DHT were non-significant after the change in skin temperature was taken into account. Intrathecal mCPP (5 micrograms) significantly increased the tail-flick latency and significantly reduced the tail skin temperature in 5,6-DHT pretreated animals when compared to control animals. These differences in tail-flick latencies could not be explained by alterations in skin temperature. The results provide evidence that neurotoxic lesion of descending 5-HT pathways induces supersensitivity to the antinociceptive effect of 5-HT1 receptor stimulation.

Acute and long term effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in tests of nociception in mice.

Acute and long term changes in nociception after administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) 80 mg/kg (four injections of 20 mg/kg given at two hr intervals) were investigated in mice. MPTP caused shivering, lacrimation, salivation, teeth chattering and fur erection a few minutes after drug injection, but all these behavioural changes were normalized within 30 min., when the first behavioural testing was performed. No significant alteration in general behaviour, sensorimotor performance or body temperature could be detected at the time of nociceptive testing. The acute effects of MPTP on nociception were a reduced response latency in the tail flick test and a prolonged response latency compared to controls in the constant temperature hot plate test. No significant effects of MPTP were found in the increasing temperature hot plate test. The long term effects were a reduced response latency both in the tail flick test and the constant temperature hot plate test, indicating that the MPTP induced lesions of dopaminergic pathways result in hyperalgesia. In the increasing temperature hot plate test and the formalin test, no significant long term changes were demonstrated. Seven days after injection, the dopamine content was reduced to 62% of control values in striatum, to 51% in the rest of the forebrain, and to 41% in the spinal cord. Noradrenaline levels were only slightly and transiently reduced. Serotonin levels were not affected 7 days after injection, but 14 days after injection, a great increase was found in the forebrain and in the spinal cord. The results suggest that dopaminergic systems tonically inhibit nociception.(ABSTRACT TRUNCATED AT 250 WORDS)

Pharmacological manipulation with the descending serotonergic system or transection of the mouse spinal cord has no effect on ependymal ultrastructure.

In order to investigate an effect of descending nerve fibres on mouse spinal cord ependymal ultrastructure, pharmacological manipulation with the serotonergic system or transection of the spinal cord was done. Biochemical analysis showed an 83% reduction of serotonin content in spinal cord tissue after p-chlorophenylalanine injections and a 93% reduction after transection. However, none of the experimental animals showed changes in ependymal ultrastructure compared to control animals as revealed by electron microscopy.

Increased behavioural response to intrathecal substance P after intracerebroventricular 5,7-dihydroxytryptamine but not after p-chlorophenylalanine administration.

The behavioural response to intrathecally injected substance P (SP, 1.25 ng) was investigated in mice after lesioning of serotonergic (5-HT) pathways by intracerebroventricular 5,7-dihydroxytryptamine (5,7-DHT, 80 micrograms base/mouse) and after 5-HT synthesis inhibition by p-chlorophenylalanine (PCPA, 400 mg kg-1 for 6 consecutive days). Pretreatment with 5,7-DHT and PCPA reduced the 5-HT level in the spinal cord to 6 and 7% of controls and the noradenaline (NA) level to 69 and 84% of controls, respectively. Intrathecally injected SP produced a response consisting of vigorous biting, licking and scratching of the caudal part of the body. The response to SP was significantly increased 5 days after injection of 5,7-DHT, but only a non-significant tendency towards enhancement of the response was found after 24 h. There was no change in the response to SP 24 h after the last injection of PCPA. It is suggested that 5,7-DHT but not PCPA induces receptor supersensitivity to SP, and that reduction in spinal SP by 5,7-DHT may be a factor in this change in receptor sensitivity.

5-HT depletion with 5,7-DHT, PCA and PCPA in mice: differential effects on the sensitivity to 5-MeODMT, 8-OH-DPAT and 5-HTP as measured by two nociceptive tests.

Depletion of 5-hydroxytryptamine (5-HT) in mice was produced by intracerebroventricular injection of 5,7-dihydroxytryptamine (5,7-DHT, 80 micrograms) or by systemic injections of p-chloroamphetamine (PCA, 3 X 40 or 4 X 40 mg/kg), p-chlorophenylalanine (PCPA, 5 X 400 or 14 X 400 mg/kg) or combined PCA (3 X 40 mg/kg) + PCPA (11 X 400 mg/kg). Neither of the pretreatments altered nociception in the increasing temperature hot-plate test, whereas hyperalgesia was demonstrated in 5,7-DHT lesioned animals in the tail-flick test. 5,7-DHT-pretreatment enhanced the antinociceptive effect of the 5-HT agonists 5-methoxy-N,N-dimethyltryptamine (5-MeODMT), 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) and 5-hydroxytryptophan (5-HTP). This effect was observed after 2, 5 and 8 days in the tail-flick test and after 5 and 8 days in the hot-plate test. However, pretreatment with PCPA or PCA failed to alter the antinociception elicited by the 5-HT agonists, although a tendency towards enhancement of antinociception was found after combined treatment with PCA and PCPA. It is suggested that the injection of 5,7-DHT induces denervation supersensitivity of post-synaptic 5-HT receptors. The lack of such supersensitivity after PCPA-pretreatment which induces similar 5-HT depletion to 5,7-DHT, may suggest that other factors than the absence of 5-HT may contribute to the development of denervation supersensitivity. Alternatively, the three 5-HT depleting agents may produce a qualitatively different reduction of 5-HT.

Involvement of central serotonergic pathways in nefopam-induced antinociception.

The possible involvement of central serotonergic pathways in the mechanism of action of nefopam was investigated in male albino mice. Nefopam (15 mg/kg i.p.) did not alter the concentration of serotonin or its metabolite 5-hydroxyindole acetic acid in frontal cortex or spinal cord. Lesions of the ascending serotonergic pathways were made by systemic administration of p-chloroamphetamine (PCA). Serotonin depletion in all serotonergic systems was obtained by means of p-chlorophenylalanine (PCPA). Two different nociceptive assays were used, the formalin test and the increasing temperature hot plate test. PCPA pretreatment significantly reduced the effect of nefopam (15 mg/kg) in the formalin test. In contrast, nefopam-induced analgesia was not affected by PCA pretreatment, either in the formalin test or in the increasing temperature hot plate test. In conclusion, the data suggest that descending serotonergic pathways are involved in nefopam-induced antinociception.

The effect of high pressure on the kinetics of monoamine transmitters in the rat brain.

The effects of 71 ATA pressure on the turnover of serotonin (5-HT), noradrenaline (NA), and dopamine (DA) were studied. High pressure increased the concentration of the 5-HT metabolite 5-hydroxyindoleacetic acid (5-HIAA) by 40%. A concomitant increase in 5-HT synthesis (measured by the accumulation of 5-hydroxytryptophan after inhibition of the decarboxylase) of 25% was found. The changes in NA and DA metabolites and synthesis were smaller and not statistically significant. The increase in 5-HT synthesis was not a result of changes in the neuronal uptake of transmitter. The observed change might be the result of a compensatory increase in the activity of the serotoninergic neurons at high pressure. Among the three monoamine transmitter systems, that mediated by 5-HT may be most important in the pathogenesis of the high pressure neurologic syndrome.

Acute effects of nomifensine on in vivo uptake and metabolism of dopamine, noradrenaline and serotonin in the rat brain.

Nomifensine, in contrast to all other antidepressants, inhibits the neuronal uptake of dopamine. The effect of this drug (10 mg/kg intraperitoneally) on the metabolism of dopamine, serotonin and noradrenaline was studied in the rat brain. After 1.5 hours, nomifensine increased the concentrations of homovanillic acid (HVA) in corpus striatum and total (free and conjugated) 3-methoxy, 4-hydroxyphenylglycol (MOPEG) in mesencephalon, but had no effect on the 5-hydroxyindoleacetic acid (5-HIAA) in pons/medulla oblongata and mesencephalon. The effect was identical with that of desipramine (25 mg/kg) on MOPEG and of imipramine (25 mg/kg) on 5-HIAA. Two methods ordinarily used for estimating turnover rates of monoamines were compared: accumulation of acid metabolites after probenecid (measuring efflux of metabolites from the brain) and accumulation of monoamine precursors after decarboxylase inhibition (measuring amine synthesis). The efflux was reduced for 5-HIAA and MOPEG but increased for HVA after nomifensine. Imipramine had the same effect on 5-HIAA and desipramine on MOPEG. Desipramine decreased the efflux of HVA from corpus striatum. In contrast, nomifensine did not change the synthesis of noradrenaline and serotonin significantly. Imipramine reduced the synthesis of serotonin in pons/medulla oblongata. In corpus striatum nomifensine, unlike imipramine, increased the concentration of 5-HIAA and synthesis of serotonin in spite of a decrease in efflux, probably because of a secondary effect from the dopaminergic action. The conclusion was made that there were more than one compartment of monoamine metabolites. The antidepressants could to some extent lead to a shift in the metabolism to sites more distant to the transport mechanism.

Lesions of the ascending serotonergic pathways and antinociceptive effects after systemic administration of p-chloroamphetamine in mice.

The present study reports a method for lesioning of the ascending serotonergic system. The neurotoxic substance p-chloroamphetamine (PCA) was given IP on 2 consecutive days (40 mg/kg/day). After each injection, the animals were kept at 4 degrees C for 4 hr since a lower dose of PCA (25 mg/kg) induced severe hyperthermia. The mortality rate was 12%, considerably lower than previously reported in similar studies. Evaluated 9 days after the last injection of PCA, the uptake of 14C-5-HT into cortical and hippocampal crude synaptosomal preparations was reduced by 50 and 60%, respectively, while the uptake into spinal synaptosomes was unaffected. The uptake of 3H-NA was not significantly altered in any of the structures. Measurements of PCA performed 30 min to 4 hr after IP injections of 5 to 40 mg/kg demonstrated higher concentrations in the cortex than in the lumbar spinal cord. Administration of PCA (5 mg/kg) had an acute antinociceptive effect in the hot-plate and formalin tests, but not in the tail-flick test. Prior treatment with neurotoxic doses of PCA prevented the antinociception but had in itself no effect on the responsiveness in any of the tests. Thus systemic administration of PCA produces highly selective and functional lesions of the ascending serotonergic pathways in mice.

Regional distribution of homocysteine in the mammalian brain.

The regional distribution of L-homocysteine (Hcy) was determined in brains from mouse, rat, guinea pig, and rabbit, using a sensitive radioenzymatic assay. Large interspecies variations in the Hcy content in various parts of the brain were observed, but cerebellum contained the highest amount in all species investigated. In the rat the amount of Hcy in cerebellum (6.4 nmol/g) was about sixfold higher than in most other parts of the brain, whereas in the mouse and guinea pig the amount in cerebellum (about 1 nmol/g) was only twofold higher than in the other brain regions. There was a remarkably high level of Hcy in all regions of the rabbit brain (4-10 nmol/g); the highest concentration was found in the cerebellar white matter. In this species the amount of Hcy in all brain regions examined exceeded that in the liver.

Homocysteine in tissues of the mouse and rat.

A method for the determination of L-homocysteine (Hcy) in tissues is described, which involves adsorption of adenosine and S-adenosyl-L-homocysteine (AdoHcy) in the tissue extract to dextran-coated charcoal, while leaving Hcy in solution. Sufficient dilution of the tissue homogenates and the presence of a reducing agent during the adsorption step are required to obtain high recovery of Hcy. Hcy is condensed with radioactive adenosine, and labeled AdoHcy is quantified by high performance liquid chromatography on a 3-micron reversed phase column. The amount of Hcy was determined in several tissues (liver, kidney, brain, heart, lung, and spleen) of mice and rats, and the concentrations of Hcy were in the range 0.5-6 nmol/g, wet weight. Hcy concentration was about 1 microM in mouse plasma. In mice, liver contained the highest amount of Hcy, and kidneys were also rich in Hcy. Similar concentrations were found in rat tissues. S-Adenosylhomocysteine (AdoHcy) hydrolase (EC 3.3.1.1), the enzyme which is believed to catalyze the only pathway leading to Hcy formation in vertebrates, was nearly completely inactivated in mice injected with the drug combination 9-beta-D-arabinofuranosyladenine plus 2'-deoxycoformycin. This treatment induced a massive accumulation of AdoHcy in all tissues (Helland, S., and Ueland, P. M. (1983) Cancer Res. 43, 1847-1850). The amount of Hcy increased several-fold in kidney, whereas no change was observed in liver, heart, brain, lung, and spleen.

Neurotoxicity of deoxycoformycin: effect of constant infusion on adenosine deaminase, adenosine, 2'-deoxyadenosine and monoamines in the mouse brain.

The tight-binding adenosine deaminase inhibitor, 2'-deoxycoformycin (dCF), was continuously infused into mice by intraperitoneal implantation of microosmotic pumps delivering the compound at a rate of 0.16 mg hr-1 kg-1 for up to 6 days. The activity of cerebral adenosine deaminase was nearly totally inhibited. The amount of adenosine and 2'-deoxyadenosine was determined in the brain frozen in liquid nitrogen through the intact skull bone. The concentration of adenosine was about 1 nmol/g, and was essentially not altered following treatment with deoxycoformycin. Deoxycoformycin induced a progressive increase in cerebral content of 2'-deoxyadenosine, which after 1 day of treatment equalled the amount of adenosine. The concentrations of serotonin, dopamine and noradrenaline in the brain were not altered.

Regional and subcellular distribution of S-adenosylhomocysteine hydrolase in the adult rat brain.

The regional distribution of S-adenosylhomocysteine hydrolase was determined in the rat brain. Small variations in enzyme activity between different regions were observed. Highest activity was was found in hypothalamus and bulbus olfactorius, the least in pons and medulla. About 70% of the enzyme activity was recovered on the soluble fraction of the tissue homogenate and 25% was localized to the crude mitochondrial fraction. The corresponding values for lactate dehydrogenase were 40% and 50%, respectively. The small amount of enzyme (5%) sedimenting with the nuclear fraction could be explained by contamination of this fraction with soluble proteins and synaptosomes. Further separation of the crude mitochondrial fraction by discontinuous sucrose gradient centrifugation showed that most of the enzyme activity was localized to the synaptosomes, but a substantial amount was found in the top layer of the gradient. The relative specific activity of lactate dehydrogenase in the top layer was less than that of S-adenosylhomocysteine hydrolase. No time-dependent leakage of S-adenosylhomocysteine hydrolase from the synaptosomes could be demonstrated. After hypoosmotic treatment of the crude mitochondrial fraction and separation of this fraction on a discontinuous sucrose gradient, S-adenosylhomocysteine hydrolase and DOPA decarboxylase showed the same distribution in the gradient and were recovered in the cytoplasmic fraction.