Early social isolation differentially affects the glucocorticoid receptor system and alcohol-seeking behavior in male and female Marchigian Sardinian alcohol-preferring rats.

Adverse early life experiences during postnatal development can evoke long-lasting neurobiological changes in stress systems, thereby affecting subsequent behaviors including propensity to develop alcohol use disorder. Here, we exposed genetically selected male and female Marchigian Sardinian alcohol-preferring (msP) and Wistar rats to mild, repeated social deprivation from postnatal day 14 (PND14) to PND21 and investigated the effect of the early social isolation (ESI) on the glucocorticoid receptor (GR) system and on the propensity to drink and seek alcohol in adulthood. We found that ESI resulted in higher levels of GR gene and protein expression in the prefrontal cortex (PFC) in male but not female msP rats. In female Wistars, ESI resulted in significant downregulation of Nr3c1 mRNA levels and lower GR protein levels. In male and female msP rats, plasma corticosterone levels on PND35 were similar and unaffected by ESI. Wistar females exhibited higher levels of corticosterone compared with males, independently from ESI. In alcohol self-administration experiments we found that the pharmacological stressor yohimbine (0.0, 0.312, 0.625, and 1.25 mg/kg) increased alcohol self-administration in both rat lines, regardless of ESI. After extinction, 0.625 mg/kg yohimbine significantly reinstated alcohol seeking in female rats only. ESI enhanced reinstatement in female msP rats. Overall, the present results indicate that repeated social deprivation during the third week of postnatal life affects GR expression in a strain- and sex-dependent manner: such effect may contribute, at least partially, to the heightened sensitivity of female msP rats to the effects of yohimbine-induced alcohol seeking.

Pin1 contribution to Alzheimer's disease: transcriptional and epigenetic mechanisms in patients with late-onset Alzheimer's disease.

BACKGROUND: Neurofibrillary tangles and senile plaques are hallmarks of Alzheimer's disease (AD) although the molecular basis of their coexistence remains elusive. The peptidyl-prolyl cis/trans isomerase Pin1 acts on both tau and amyloid precursor protein to regulate their functions by influencing tau phosphorylation and amyloid precursor protein processing. OBJECTIVE: In order to identify potential biomarkers for AD in easily accessible cells and to gain insight into the relationship between the brain and peripheral compartments in AD pathology, we investigated Pin1 expression and activity in the peripheral blood mononuclear cells of subjects with late-onset AD (LOAD) and age-matched controls (CT). METHODS: Gene and protein expression, promoter methylation, Ser(16) phosphorylation and activity of Pin1 were evaluated in 32 samples from subjects with LOAD and in 28 samples from CT. RESULTS: In LOAD subjects, there was a statistically significant reduction in Ser(16) phosphorylation (-30%; p = 0.041) and promoter methylation (-8%; p = 0.001), whereas Pin1 expression was significantly increased (+74%; p = 0.018). CONCLUSION: The modifications of Pin1 found in LOAD subjects support its involvement in the pathogenesis of the disease with an important role being played by epigenetic mechanisms.

Dynorphinergic system alterations in the corticostriatal circuitry of neuropathic mice support its role in the negative affective component of pain.

The dynorphinergic system is involved in pain transmission at spinal level, where dynorphin exerts antinociceptive or pronociceptive effects, based on its opioid or non-opioid actions. Surprisingly, little evidence is currently available concerning the supraspinal role of the dynorphinergic system in pain conditions. The present study aimed to investigate whether neuropathic pain is accompanied by prodynorphin (Pdyn) and κ-opioid receptor (Oprk1) gene expression alterations in selected mouse brain areas. To this end, mice were subjected to chronic constriction injury of the right sciatic nerve and neuropathic pain behavioral signs were ascertained after 14 days. At this interval, a marked increase in Pdyn mRNA in the anterior cingulate cortex (ACC) and prefrontal cortex (PFC) was observed. Oprk1 gene expression was increased in the PFC, and decreased in the ACC and nucleus accumbens (NAc). No changes were observed in the other investigated regions. Because of the relationship between dynorphin and the brain-derived neurotrophic factor, and the role of this neurotrophin in chronic pain-related neuroplasticity, we investigated brain-derived neurotrophic factor gene (Bdnf) expression in the areas showing Pdyn or Oprk1 mRNAs changes. Bdnf mRNA levels were increased in both the ACC and PFC, whereas no changes were assessed in the NAc. Present data indicate that the dynorphinergic system undergoes quite selective alterations involving the corticostriatal circuitry during neuropathic pain, suggesting a contribution to the negative affective component of pain. Moreover, parallel increases in Pdyn and Bdnf mRNA at cortical level suggest the occurrence of likely interactions between these systems in neuropathic pain maladaptive neuroplasticity.

Role of serotonin in the regulation of the dynorphinergic system by a kappa-opioid agonist and cocaine treatment in rat CNS.

It has been shown that chronic cocaine increases prodynorphin mRNA in the caudate putamen and decreases it in the hypothalamus. In addition, treatment with a kappa-opioid receptor agonist produced the opposite effect on prodynorphin gene expression in these brain regions and also evoked a decrease in the hippocampus. It is already known that kappa-opioid receptor agonists decrease the development of sensitization to some of the behavioral effects of cocaine. The serotonin system has also been shown to regulate dynorphin gene expression and a continuous infusion of fluoxetine induced prodynorphin gene expression in the same pattern as the kappa-opioid agonist (+)(5a,7a,8b)-N-methyl-N-[7-(1-pyrrolidinyl)-1 oxaspiro[4.5]dec-8-yl]-benzeneacetamide (U-69593) in the brain regions investigated. It is interesting to note that treatment with a continuous infusion of cocaine produced different effects on this parameter. To determine whether serotonin plays a role in the regulation of prodynorphin mRNA by kappa-opioid agonists or cocaine, rats were treated with the serotonin depleter parachloroamphetamine (PCA). Beginning 24 h later, rats were treated with the selective kappa-opioid agonist U-69593 for 5 days or continuously with cocaine for 7 days and prodynorphin mRNA was measured. Prodynorphin mRNA was decreased significantly in the hypothalamus, caudate putamen, and hippocampus of rats treated with a single injection of PCA. Subsequent to PCA administration the effects of U-69593 or cocaine on prodynorphin mRNA were differentially affected across brain regions. Prodynorphin gene expression was still increased by U-69593 treatment in the hypothalamus and decreased in the caudate putamen. Cocaine treatment still produced a decrease in this parameter in the hypothalamus and an increase in the caudate putamen. In contrast, in the hippocampus, the decrease in prodynorphin mRNA produced by U-69593 was no longer evident after PCA and cocaine, which previously had no effect, now increased it in the serotonin-depleted group. These findings suggest that serotonin is necessary to maintain normal levels of dynorphin mRNA in all of the investigated brain areas and that the regulation of prodynorphin mRNA expression by chronic treatment with a kappa-opioid receptor agonist or cocaine requires serotonin in the hippocampus, but not in the hypothalamus or caudate putamen.

N/OFQ system in brain areas of nerve-injured mice: its role in different aspects of neuropathic pain.

Several studies showed that chronic pain causes reorganization and functional alterations of supraspinal brain regions. The nociceptin-NOP receptor system is one of the major systems involved in pain control and much evidence also suggested its implication in stress, anxiety and depression. Therefore, we investigated the nociceptin-NOP system alterations in selected brain regions in a neuropathic pain murine model. Fourteen days after the common sciatic nerve ligature, polymerase chain reaction (PCR) analysis indicated a significant decrease of pronociceptin and NOP receptor mRNA levels in the thalamus; these alterations could contribute to the decrease of the thalamic inhibitory function reported in neuropathic pain condition. Nociceptin peptide and NOP mRNA increased in the anterior cingulate cortex (ACC) and not in the somatosensory cortex, suggesting a peculiar involvement of this system in pain regulating circuitry. Similarly to the ACC, an increase of nociceptin peptide levels was observed in the amygdala. Finally, the pronociceptin and NOP mRNAs decrease observed in the hypothalamus reflects the lack of hypothalamus-pituitary-adrenal axis activation, already reported in neuropathic pain models. Our data indicate that neuropathic pain conditions affect the supraspinal nociceptin-NOP system which is also altered in regions known to play a role in emotional aspects of pain.

Chronic and acute effects of 3,4-methylenedioxy-N-methylamphetamine ('Ecstasy') administration on the dynorphinergic system in the rat brain.

The prodynorphin system is implicated in the neurochemical mechanism of psychostimulants. Exposure to different drugs of abuse can induce neuroadaptations in the brain and affect opioid gene expression. The present study aims to examine the possibility of a common neurobiological substrate in drug addiction processes. We studied the effects of single and repeated 3,4-methylenedioxy-N-methylamphetamine ('Ecstasy') on the gene expression of the opioid precursor prodynorphin, and on the levels of peptide dynorphin A in the rat brain. Acute (8 mg/kg, intraperitoneally) 3,4-methylenedioxy-N-methylamphetamine markedly raised, two hours later, prodynorphin mRNA levels in the prefrontal cortex, and in the caudate putamen, whereas it decreased gene expression in the ventral tegmental area. Chronic (8 mg/kg, intraperitoneally, twice a day for 7 days) 3,4-methylenedioxy-N-methylamphetamine increased prodynorphin mRNA in the nucleus accumbens, hypothalamus and caudate putamen and decreased it in the ventral tegmental area. Dynorphin A levels increased after chronic treatment in the ventral tegmental area and decreased after acute treatment in the nucleus accumbens, prefrontal cortex and hypothalamus. These findings confirm the role of the dynorphinergic system in mediating the effects of drugs of abuse, such as 3,4-methylenedioxy-N-methylamphetamine, in various regions of the rat brain, which may be important sites for the opioidergic mechanisms activated by addictive drugs.

Alterations in prodynorphin gene expression and dynorphin levels in different brain regions after chronic administration of 14-methoxymetopon and oxycodone-6-oxime.

Previous studies showed that opioid drugs-oxycodone-6-oxime and 14-methoxy-5-methyl-dihydromorphinone (14-methoxymetopon)-produced less respiratory depressive effect and slower rate of tolerance and dependence, respectively. It was also reported that morphine decreased the prodynorphin gene expression in the rat hippocampus, striatum and hypothalamus. In this study, we determined the prodynorphin gene expression and dynorphin levels in selected brain regions of opioid tolerant rats. We found that in the striatum morphine decreased, while oxycodone-6-oxime increased and 14-methoxymetopon did not alter the prodynorphin gene expression. In the nucleus accumbens, morphine and oxycodone-6-oxime did not change, while 14-methoxymetopon increased the prodynorphin gene expression. In the hippocampus both oxycodone-6-oxime and 14-methoxymetopon enhanced, whereas morphine did not alter the prodynorphin gene expression. In the rat striatum only oxycodone-6-oxime increased dynorphin levels significantly in accordance with the prodynorphin mRNA changes. In the hippocampus both opioid agonists increased the dynorphin levels significantly similarly to the augmented prodynorphin gene expression. In ventral tegmental area only 14-methoxymetopon increased dynorphin levels significantly. In nucleus accumbens and the temporal-parietal cortex the changes in the prodynorphin gene expression and the dynorphin levels did not correlate. Since the endogenous prodynorphin system may play a modulatory role in the development of opioid tolerance, the elevated supraspinal dynorphin levels appear to be partly responsible for the reduced degree of tolerance induced by the investigated opioids.

Effects of acute ethanol exposure on class I HDACs family enzymes in wild-type and BDNF(+/-) mice.

BACKGROUND: Alterations of brain-derived neurotrophic factor (BDNF) have been associated with the development of addiction to different drugs of abuse, including ethanol (EtOH). EtOH exposure activates the BDNF-signaling cascade in dorsal striatum, which in turn affects further EtOH intake. Different alcohol exposures have been widely demonstrated to modulate chromatin remodeling, affecting histone acetylation/deacetylation balance. Recently, class I histone deacetylases (HDACs) inhibition has been reported to modulate BDNF mRNA expression and to attenuate morphological and behavioral phenomena related to EtOH exposure. However, the role played by different HDAC isoforms in EtOH-induced plasticity is still unclear. METHODS: We investigated the effects induced by acute EtOH exposure on the protein levels of class I HDAC 1-3 isoforms of wild-type (WT) and BDNF heterozygous mice (BDNF(+/-)), in nuclear and cytoplasmic extracts of specific brain regions associated with EtOH addiction. RESULTS: Nuclear HDAC 1-3 levels were markedly reduced after acute EtOH treatment in the caudate putamen (CPu) of WT mice only. Furthermore, CPu basal levels of nuclear HDAC isoforms were significantly lower in BDNF(+/-) mice compared to WT. With the exception of nuclear HDAC 3, no significant changes were observed after acute EtOH treatment in the prefrontal cortex (PFCx) of BDNF(+/-) and WT mice. In this area, the nuclear HDAC basal levels were significantly different between the two experimental groups. CONCLUSIONS: These results provide details about EtOH effects on class I HDAC isoforms and strongly support a correlation between BDNF and class I HDACs, suggesting a possible influence of BNDF on these enzymes.

The effect of a paracetamol and morphine combination on dynorphin A levels in the rat brain.

The purpose of this study was to find out whether the combination of inactive doses of paracetamol (PARA) and morphine was able to change dynorphin (DYN) A levels, evaluated by radioimmunoassay, and whether naloxone or [(-)-2-(3 furylmethyl)-normetazocine] (MR 2266), a kappa-opioid antagonist, modifies or prevents the activity of this combination on nociception and on DYN levels. The work was suggested by our previous findings which demonstrated that inactive doses of PARA and morphine, when given in combination, share an antinociceptive effect, and that PARA, at antinociceptive doses, decreases DYN levels in the frontal cortex, thus indicating a selective action within the CNS. Our present results demonstrate that the combination of inactive doses of PARA (100 mg/kg) and morphine (3 mg/kg) is just as effective in decreasing the levels of DYN A as full antinociceptive doses of PARA or morphine alone in the frontal cortex of the rat. The values, expressed in pmol/g tissue, were: control = 2.83 +/- 0.20; paracetamol (100) = 2.60 +/- 0.23; morphine (3) = 2.73 +/- 0.24; paracetamol + morphine = 1.34 + 0.16 (P < 0.05). The decrease was partially antagonised by MR 2266, but not by naloxone, suggesting that the activity of PARA and morphine in combination on DYN A levels could be mediated, at least in part, through kappa-receptors, although other systems may be involved. On the other hand, both naloxone and MR 2266 prevented the antinociceptive effect of the combination in the hot plate test. All our experimental data suggest that PARA and morphine in combination exert their antinociceptive effect through the opioidergic system, which in turn may cause a decrease in DYN levels in the CNS of the rat.

Limbic seizures increase pronociceptin mRNA levels in the thalamic reticular nucleus.

We studied pronociceptin gene expression following limbic seizures. Northern blot analysis revealed increased pronociceptin mRNA levels in the thalamus (but not in the hippocampus) 3-24 h after kainate administration, with maximal effect (2-fold increase over basal levels) reached at 6 h. No variation in pronociceptin mRNA levels was observed 1-6 h after a stimulus-evoked kindled seizure. Carrageenan failed to affect pronociceptin gene expression in the thalamus, indicating that pain and/or acute stress do not account for kainate effects. In situ hybridization revealed that kainate evokes a dramatic (4-fold) increase in pronociceptin mRNA levels over the thalamic reticular nucleus. Kindled seizures evoked only a small, non-significant increase in pronociceptin gene expression over the dentate gyrus of the hippocampus.

Effects of an antisense oligonucleotide to pronociceptin and long-term prevention of morphine actions by nociceptin.

To further characterize the anti-opioid action of the neuropeptide nociceptin, we examined the effects of the repeated intracerebroventricular (i.c.v.) treatment (once daily for 4 days) with an antisense oligodeoxynucleotide complementary to pronociceptin mRNA, in the rat. We also investigated possible changes of the antinociceptive and hyperthermic effects induced by the i.c.v. administration of morphine, in rats i.c.v. pretreated with nociceptin 3 h before. The pretreatment with the antisense oligodeoxynucleotide, but not with a mismatched sequence (used as a control), caused an increase in spontaneous locomotor activity and produced a potentiation of the antinociceptive effect of a submaximal dose of i.c.v. morphine (1 microgram/rat). The i.c.v. pretreatment with nociceptin (2 nmol/rat, 3 h before) prevented both the antinociceptive and the hyperthermic effects of morphine (10 microgram/rat i.c.v.). These results strengthen the hypothesis of an anti-opioid action of nociceptin at supraspinal level and suggest that the neuropeptide may exert long-term modulatory effects.

Studies on the antinociceptive effect of intrathecal salmon calcitonin.

The involvement of spinal opioid receptors and spinal monoaminergic systems, in the antinociceptive effect of intrathecal (IT) salmon calcitonin, has been evaluated by means of the hot plate test, in the rat. Intrathecal pretreatment with 40 micrograms MR 1452 and 40 micrograms ICI 154,129, purported selective antagonists respectively for kappa and delta opioid receptors did not modify sCT-induced antinociception (2 micrograms IT). A delay in the development of IT sCT-induced antinociception was observed in rats selectively depleted of cord serotonin (25 mg/kg IP desipramine plus 100 micrograms IT 5,7-dihydroxytryptamine), whereas the administration of serotonergic antagonists, methysergide and ketanserin, 30 micrograms IT, did not influence sCT effect. Cord catecholamine depletion (6-OHDA pretreatment) reduced significantly sCT antinociception. A similar reduction was produced by the dopaminergic antagonist haloperidol (15 micrograms IT), but not by the alpha-blocker phentolamine (15 micrograms IT). Findings of this study rule out an involvement of opioid peptidergic system in sCT-induced increase of hot plate latencies at spinal level; a possible involvement of cord dopaminergic receptors is suggested.

Antinociceptive profile of intracerebroventricular salmon calcitonin and calcitonin gene-related peptide in the mouse formalin test.

The effects of intracerebroventricularly administered salmon calcitonin (sCT) and calcitonin gene-related peptide (CGRP) on the behavioural response of the mouse to formalin injections were investigated. Mice lick their hindpaws for 5 min after formalin injections, then stop, and resume intensive licking for another 10 min beginning 20 min after the injections. Both peptides reduced the nociceptive response in the two phases of the test (0-5 and 20-30 min after formalin injection). Antinociceptive A50 values were 3.3 micrograms/mouse and 4.7 micrograms/mouse respectively in the first phase for sCT and CGRP. The effects of sCT and CGRP appeared to be dose-dependent in the first phase. Since in the second phase sCT appeared more effective and CGRP gave a bell-shaped curve, possible differences in the mechanisms of action of the peptides in the two response intervals of the test are suggested.

Hypothermia elicited by some prodynorphin-derived peptides: opioid and non-opioid actions.

Prodynorphin-derived peptides were tested for their effects on body temperature after intracerebroventricular administration to unrestrained male rats. Dynorphin A (Dyn A) (5 and 10 nmol) and Dynorphin A-(1-32) (Dyn A-(1-32) (2.5 and 5 nmol) lowered body temperature with a maximum approximately 30 min after administration. Dyn B (up to 50 nmol) did not induce hypothermia. Lower doses of all peptides did not alter body temperature. The hypothermic effect was significantly, but not completely prevented by MR1452 (30 nmol), a preferential antagonist of the kappa receptor, administered intracerebroventricularly. Naloxone, a mu receptor antagonist, naltrexone, its long acting analog up to doses of 100 nmol, as well as MR1453, the (+)-enantiomer of kappa antagonist MR1452 with no opioid binding properties, did not prevent the hypothermic effect. Moreover, episodic barrel rolling and bizarre postures elicited by Dyn A and Dyn A-(1-32) were reduced in rats pretreated i.c.v. with MR1452 (30 nmol), but not with naloxone (up to 100 nmol). Interestingly, des-Tyr-Dynorphin A (Dyn A-(2-17)), a fragment with virtually no opioid binding potential, was 4 times less potent that Dyn A in inducing hypothermia. These findings are consistent with the hypothesis that prodynorphin-derived peptides effects are not exclusively opioids in nature.

Relationship of analgesia induced by centrally injected calcitonin to the CNS serotonergic system.

The role of the serotonergic system in the antinociceptive effect of centrally administered salmon calcitonin (sCT) was studied in rats. The animals were given sCT either intracerebroventricularly (i.c.v.) or intrathecally (i.t.). I.c.v. administration of sCT (2,5 micrograms/rat) to animals depleted in CNS serotonin (5-HT) either by treatment with 25 mg/kg desmethylimipramine (DMI) i.p. plus 100 micrograms/rat 5,7 dihydroxytryptamine (5,7 DHT) i.c.v., ten days before or by 150 mg/kg p-chlorophenylalanine (pCPA) i.p., 72 and 24 h before, still significantly increased the hot-plate latencies comparable to those of non-depleted animals. The same result was obtained when the 5-HT receptors were blocked with metergoline. The i.t. administration of sCT (2 micrograms/rat) to animals with spinal cord 5-HT depleted by treatment with DMI plus 5,7 DHT, i.t., delayed but did not abolish the antinociceptive activity of i.t. injected sCT, which was of the same intensity as in non depleted animals. When 5,7 DHT was administered alone, either i.c.v. or i.t., without protection of the catecholaminergic neurons so that noradrenaline (NA) was greatly reduced, the antinociceptive effect of sCT was completely abolished even when NA had been depleted only in the spinal cord. We conclude that it is the catecholaminergic system, not the serotonergic, that plays a fundamental role in the anti-nociceptive effect of centrally administered sCT.

Possible involvement of dynorphinergic system in nociceptive transmission at spinal level.

The opioid peptide dynorphin1-32 (DYN1-32, 25 nmol) intrathecally administered causes, in the rat, an elevation of nociceptive threshold of longer duration than that of DYN A, as ascertained by vocalization test. Comparative findings obtained with tail flick test allow to differentiate antinociception from motor dysfunction. The breakdown of DYN A at spinal level is very rapid. The electrical stimulation of the tail associated to a restraint condition of the rat produces a significant increase of immunoreactive DYN in cervical, thoracic and lumbar segments of spinal cord, therefore indicating a correlative, if not causal, relationship between the spinal dynorphinergic system and aversive stimuli.

The effect of paracetamol on nociception and dynorphin A levels in the rat brain.

Male Wistar rats were administered with naloxone (1 mg/kg i.p.) or MR 2266 (5 mg/kg i.p) 15 min before paracetamol (400 mg/kg i.p.) treatment and the pain threshold was evaluated. Rats were subjected to the hot-plate and formalin tests and immunoreactive dynorphin A (ir-dynorphin A) levels were measured in the hypothalamus, hippocampus, striatum, brainstem, frontal and parietal-temporal cortex by radioimmunoassay. Pretreatment with naloxone abolished paracetamol antinociceptive activity both in hot-plate and in the first phase, but not in the second phase of the formalin test, while MR 2266 pretreatment was able to antagonise paracetamol effect either in the hot-plate test or in both phases of the formalin test. Among different brain areas investigated paracetamol significantly decreased ir-dynorphin A levels only in the frontal cortex. MR 2266 but not naloxone reversed the decrease in ir-dynorphin A levels elicited by paracetamol. Paracetamol seems to exert its antinociceptive effect also through the opioidergic system modulating dynorphin release in the central nervous system (CNS) of the rat, as suggested by the decrease in the peptide levels.

Regional distribution of immunoreactive dynorphin A in the human gastrointestinal tract.

Immunoreactive dynorphin A (ir-Dyn A) was detected throughout the human gastrointestinal tract by a validated radioimmunoassay. Moreover, the stability of 125I-Dyn A during extraction procedures was confirmed by high performance liquid chromatography. Levels of ir-Dyn A were higher in the stomach and in the small bowel. In tissue samples separated into the main layers composing the gut wall (muscularis externa, submucosa and mucosa) ir-Dyn A was uniformly distributed. An exception was the colon, where concentrations were higher in the muscular portion. Gel permeation chromatography on samples of mucosa and muscularis externa extracts of ileum and gastric fundus, showed immunoreactive material eluting in several forms of apparently higher molecular weight than Dyn A, while only a minor peak was found to coelute with authentic Dyn A.

Cerebrospinal alterations of immunoreactive dynorphin A after unilateral dorsal rhizotomy in the rat.

Possible alterations of immunoreactive dynorphin A (ir-dyn A) were investigated at different levels of the spinal cord and in discrete brain regions of male rats 10, 30 and 60 days after unilateral dorsal rhizotomy, i.e., during the development of deafferentation pain and autotomy behavior that follows afferent nerve interruption. Dorsal rhizotomy caused an increase of spinal ir-dyn A at 10 days in the cervical segment; subsequent assays showed a progressive increase in other spinal regions too. At the last observation, 60 days after rhizotomy, neuropeptide levels were still significantly higher than in sham-lesioned animals in the cervical, thoracic and lumbosacral spinal cord. The spinal ir-dyn A changes were both ipsi- and contralateral to the lesion. No alterations were found in the brainstem and midbrain and a not significant decrease was observed in the hypothalamus. In the striatum and cortex, however, there was a bilateral significant increase 30 days after surgery and a constant and significant elevation was detected in the hippocampus at all three intervals. These data cast additional light on the neurochemical changes caused by the interruption of afferent nerves, followed by development of the deafferentation pain syndrome in laboratory animals and human beings. They also support the concept of central neuroplasticity in pathological pain and indicate that the opioid neuropeptide dynorphin is involved.

Characterization of dynorphin A-induced antinociception at spinal level.

Dynorphin A (DYN A) injected intrathecally in the rat produced a significant elevation of the nociceptive threshold, measured by the tail flick test. The highest dose of DYN A (25 nmol) produced maximal elevation of tail flick latency to radiant heat together with hindlimb paralysis and tail flaccidity lasting several hours, thus confirming several previous reports. A lower dose of DYN A (12.5 nmol) produced only a smaller, not constant, short-lasting change in the nociceptive threshold. The vocalization test (electrical stimulation of the tail) gave a different result: the time course curve showed that the antinociceptive effect had worn off 60 min after DYN A 25 nmol. Thus it can be assumed that the prolonged depression of the tail flick reflex was related to motor dysfunction and did not completely reflect the animal's response to painful stimuli. Tolerance to the antinociceptive and motor effects developed after the chronic intrathecal infusion of DYN A with osmotic minipumps. Intrathecal MR 1452 (30 nmol), a purported kappa-receptor blocker, fully prevented the effects of DYN A but not morphine-induced antinociception. Naloxone antagonized DYN A only at a 4 fold higher dose. MR 1452 (90 nmol) administered after DYN A reversed the elevation of the vocalization threshold while tail flick latency remained unmodified. Analysis by high performance liquid chromatography of intrathecally injected radiolabelled DYN A revealed that DYN A was largely broken down about 10 min after its administration. Our results seem to indicate that DYN A in the spinal cord causes alterations in nociception and motor function, clearly distinguishable in time and both mediated by an opioid receptor, probably of the kappa type. However, different mechanism(s), possibly non-opioid in nature, may contribute to the prolonged depression of the tail flick.