J. Turchan

Morphine and cocaine influence on CRF biosynthesis in the rat central nucleus of amygdala.

The central nucleus of the amygdala is a CRF-containing limbic brain site which mediates both fear-like and avoidance behaviors; moreover it has been hypothesized that atypical stress responses may contribute to compulsive drug use. Therefore, we studied in rat amygdala the level of CRF mRNA by in situ hybrydization, and the level of the peptide using immunocytochemistry after acute and chronic administration of morphine and cocaine and after their withdrawal. Acute injection of morphine (20 mg/kg i.p.) increased CRF mRNA level, but did not change significantly CRF immunoreactivity in the central nucleus of the amygdala. Chronic morphine administration significantly increased the level of CRF mRNA 3, 24 and 48 h after the last dose. Both, acute and chronic cocaine administration increased CRF mRNA, but the peptide level was decreased only after acute cocaine administration. However, in the late withdrawal (48 h after the last dose of cocaine) both mRNA and the peptide levels tended to decrease. The above data suggest that amygdalar CRF system activity is potently activated after administration of morphine and cocaine, and that activation of this system observed at the time of withdrawal from morphine may be responsible for aversion and anxiety related to these states; therefore a CRF1 receptor may be a target for prospective pharmacotherapies of the withdrawal from abused drugs.

The effect of pentoxifiline on post-injury hyperalgesia in rats and postoperative pain in patients

Recent studies demonstrate that activation of proinflammatory cytokines following injury intensifies the process of nociception. The present investigation assessed the influence of pre-injury pentoxifiline (PTFL, a non-specific cytokine inhibitor) on the development of post-injury nociception in animals and patients. It was established that intrathecal or intraperitoneal PTFL, elevated the nociceptive threshold for mechanical stimuli in the formalin test in rats. Pre-injury PTFL also inhibited pain-related behaviour. These findings correlate with a lower TNFalpha level in the serum of animals receiving pre-injury PTFL. In clinical investigations PTFL was administered intravenously before elective cholecystectomy. Patients who received preoperative PTFL had lower opioid requirements in the early postoperative period than control. At the same time, serum levels of TNFalpha and IL6 were lower in the PTFL group. Our results confirm the hypothesis as to the possibility of modulating of nociception through preemptive administration of a cytokine inhibitor.

Ethanol withdrawal enhances the prodynorphin system activity in the rat nucleus accumbens

The present study investigated the effects of ethanol withdrawal after its chronic administration on endogenous opioid systems in the nucleus accumbens of rats. An in situ hybridization study showed an increase in the prodynorphin mRNA level at 24 and 48 h (by 189 and 146%, respectively) after ethanol withdrawal, whereas the proenkephalin mRNA level remained unchanged. Furthermore, after a 48 h withdrawal period, the level of alpha-neoendorphin (alphaNEO), a prodynorphin-derived peptide, was significantly decreased (by 48%), that effect being associated with the enhancement of the K+-stimulated release of that peptide from nucleus accumbens slices. At 96 h after ethanol withdrawal, only the basal release of alphaNEO was elevated, while other parameters returned to the control level. Our data indicate that after 48 h of ethanol withdrawal, prodynorphin neurons are highly activated. The increased supply of endogenous kappa opioid receptor agonists in the nucleus accumbens at that time may promote aversive states during ethanol withdrawal.

Effects of single and repeated morphine administration on the prodynorphin, proenkephalin and dopamine D2 receptor gene expression in the mouse brain

Effects of single (20 mg/kg i.p.) and repeated morphine administration (increasing doses: from 10 to 50 mg/kg i.p. twice daily for 7 days) on the proenkephalin (PENK), prodynorphin (PDYN) and dopamine D2 receptor (D2) mRNA levels in the nucleus accumbens and striatum of the mouse were investigated. As shown by an in situ hybridization, a single dose of morphine had no significant effect on the PDYN, PENK and D2 mRNA levels in the nucleus accumbens and striatum. Repeated treatment with morphine increased the PDYN mRNA level in both those structures after 2 and 72 h. In contrast to PDYN, the PENK mRNA level was reduced in the nucleus accumbens and remained unchanged in the striatum following repeated morphine administration. Repeated morphine had no effect on the D2 mRNA level in the nucleus accumbens and striatum after 2 h, and decreased it in the nucleus accumbens after 72 h only. The above results indicate that repeated morphine leads to long-lasting upregulation of the PDYN gene expression in the mouse nucleus accumbens and striatum; on the other hand, the PENK and D2 mRNA gene expressions are either inhibited or remain unchanged, significant changes being observed in the nucleus accumbens only.

The effect of single and repeated morphine administration on the prodynorphin system activity in the nucleus accumbens and striatum of the rat

Pharmacological data indicate that prodynorphin peptides and exogenous kappa agonists affect opioid tolerance and dependence. In order to elucidate the activity of the endogenous prodynorphin system during opiate tolerance and dependence, we investigated the effect of single and repeated morphine administration on the alpha-neoendorphin tissue level, its in vitro release, and the prodynorphin messenger RNA level in the nucleus accumbens and striatum of the rat. Acute and repeated morphine administration (14 days, increasing doses, 20-100 mg/kg, i.p.) increased the level of alpha-neoendorphin in the nucleus accumbens after 3 h; a similar effect was observed at 24 and 48 h after the last chronic morphine injection. On the other hand, the basal and stimulated (K+, 57 mM) release of alpha-neoendorphin from nucleus accumbens slices were significantly elevated only at 24 h after the last morphine injection. The prodynorphin messenger RNA hybridization signal in the nucleus accumbens was enhanced at 3 h after acute morphine injection, whereas repeated morphine administration decreased the messenger RNA level at that time point. Upon late chronic morphine withdrawal (at 24 and 48 h), the prodynorphin messenger RNA level in that tissue was significantly elevated. In the striatum, single morphine administration had no effect on the alpha-neoendorphin tissue level, release of the peptide, and prodynorphin messenger RNA level. On the other hand, chronic injection of morphine elevated all those parameters. The tissue level of alpha-neoendorphin was elevated at 3 h, and was back to normal at 24 and 48 h after the last drug injection. Both the basal and stimulated alpha-neoendorphin release from striatal slices was significantly increased at all the time points studied. Repeated morphine administration elevated the striatal prodynorphin messenger RNA level at 24 and 48 h after the drug withdrawal. Addition of morphine to the incubation medium reduced the basal release of alpha-neoendorphin in both the nucleus accumbens and striatal slices in naive animals, whereas the stimulated release was attenuated in the latter tissue only. The present study indicates that withdrawal of chronic morphine leads to enhancement of the prodynorphin neurons activity in the nucleus accumbens and striatum of the rat. It is suggested that these effects may participate in the mechanism of aversive reactions during withdrawal.

The effect of drugs of abuse on NMDAR1 receptor expression in the rat limbic system

An increasing body of evidence points to the role of N-methyl-D-aspartate (NMDA) receptors in the limbic system in the mechanism of drug dependence. We studied the influence of acute and repeated morphine (20 mg/kg i.p. or increasing dose for 10 days) and cocaine (3x20 mg/kg i.p. per day at hourly intervals, for 1 or 5 days) administration on the expression of glutamate NMDA receptor subunit 1 (NMDAR1) in the central and basolateral nuclei of the rat amygdala and hippocampal formation. Acute or chronic morphine and cocaine administration increased NMDAR1 mRNA level in the central and basolateral nuclei of the amygdala; morphine did so 3 h after the last dose and 48 h after withdrawal, cocaine 3 h after acute and last chronic dose. Morphine did not change the NMDAR1 mRNA level in the hippocampal formation, but chronic cocaine did decrease it in the dentate gyrus only. Our study suggests a possible link between the expression of NMDAR1 and changes in limbic system neuronal activity and behaviour after administration of morphine and cocaine. In summary, the present study demonstrated that morphine and cocaine influenced the expression of NMDAR1 in the structure of the limbic system which could be involved in dependence phenomena.

Effect of 6-hydroxydopamine on neuropeptide Y and corticotropin-releasing factor expression in rat amygdala.

The influence of dopaminergic denervation on neuropeptide Y and corticotropin-releasing factor-containing neurons in the amygdala was investigated in rats by examining the effects of a selective, unilateral 6-hydroxydopamine lesion of mesencephalic dopaminergic neurons in both the substantia nigra and the ventral tegmental area on these peptides and their messenger RNA expression, observed eight to 10 days after the lesion. The studies were conducted by immunocytochemical and in situ hybridization methods. Neuropeptide Y or corticotropin-releasing factor-immunoreactive neurons were counted in sections of the amygdala under a microscope, and the messenger RNA expression was measured as optical density units in autoradiograms. A significant increase in both neuropeptide Y and corticotropin-releasing factor messenger RNA expression was found in the amygdala on the lesioned side in comparison with the contralateral one, as well as with the ipsilateral side of vehicle-injected controls. Immunohistochemical studies showed that the number of neuropeptide Y-immunoreactive neurons increased in the whole amygdala on the lesioned side. At the same time, the number of corticotropin-releasing factor-immunoreactive neurons grouped in the central amygdaloid nucleus declined, and so did the staining intensity. The obtained results indicate that dopaminergic denervation stimulates the synthesis of neuropeptide Y and corticotropin-releasing factor in rat amygdala, but the peptide levels are differently regulated, which points to a diverse release of these peptides.

Effects of pentylenetetrazol kindling on glutamate receptor genes expression in the rat hippocampus.

It has been hypothesized that changes in the excitatory amino acid receptor biosynthesis may be involved in the mechanism of kindling-an animal model of epileptogenesis. In order to test this hypothesis, we investigated the effects of pentylenetetrazol kindling on the expression of genes coding for NMDAR1 and GluR2 in the rat hippocampal formation. Pentylenetetrazol kindling decreased the hippocampal NMDAR1 mRNA level after 3 and 24 h; lowered the GluR2 flip level and elevated the flop mRNA one in the CA1 field and dentate gyrus after 3 and 24 h, respectively. A receptor autoradiography showed an increase in the [3H]MK-801 binding density in the hippocampus following both acute and repeated pentylenetetrazol administration. We conclude that an early occurrence of downregulation of the glutamate receptor gene expression may be an adaptive response of glutamate receptors to an oversupply of excitatory amino acids during repeated seizures.

Pharmacological studies on the monoaminergic influence on the synthesis and expression of neuropeptide Y and corticotropin releasing factor in rat brain amygdala

Our earlier findings concerning the 6-OHDA lesion suggested dopaminergic regulation of neuropeptide Y (NPY) and corticotropin releasing factor (CRF) synthesis and expression in amygdala neurons. On the other hand, some other studies indicated that not only dopamine, but also other monoamines may modulate peptidergic neurons. Therefore the present study examined the effect of pharmacological deprivation of monoaminergic influences on NPY and CRF neurons in rat brain amygdala by means of in situ hybridization and immunohistochemical methods. It was found that NPY mRNA expression in the amygdala decreased after 24h blockade of dopaminergic D1 and D2 receptors, by haloperidol or SCH23390. At the same time the NPY-peptide expression measured immunohistochemically was not significantly changed. A prolonged, 14-day, blockade of dopaminergic receptors by haloperidol induced an opposite effect, an increase in NPY mRNA expression. Impairment of the serotonergic transmission by blockade of 5-HT synthesis using p-chlorophenylalanine, as well as attenuation of the noradrenergic transmission by NA depletion from terminals by DSP4, did not significantly change NPY mRNA expression or the mean number of NPY-immunoreactive neurons in the amygdala. Only a decrease in the staining intensity observed as a decreased number of darkly stained neurons was found after both compounds. Neither the dopamine receptor blockade nor the impairment of serotonergic or noradrenergic transmission changed CRF mRNA or the peptide expression in the amygdala. The obtained results indicate that in rat brain amygdala, of all the monoamines, dopamine seems to be the most important modulator of NPY biosynthesis and expression. The effect of blockade of dopaminergic receptors is biphasic: first it induces a decrease and then - after prolonged treatment an increase in NPY mRNA. Serotonergic and noradrenergic systems in the amygdala seem to be connected with regulation of NPY release rather than the biosynthesis.

Effects of pilocarpine- and kainate-induced seizures on thyrotropin-releasing hormone biosynthesis and receptors in the rat brain.

Summary. The expression of mRNA coding for prepro-thyrotropin releasing hormone (preproTRH) was estimated in the rat brain in two animal models of limbic seizures, evoked by systemic administration of pilocarpine (400 mg/kg ip) or kainate (12 mg/kg ip). As shown by an in situ hybridization study, after 24 h both pilocarpine- and kainate-induced seizures profoundly increased the preproTRH mRNA level in the dentate gyrus. After 72 h, the preproTRH mRNA level was back to control values. Kainate-treated rats showed an elevated level of TRH in the hippocampus, septum, frontal and occipital cortex after 24 and 72 h, whereas in the striatum and amygdala the TRH level was raised after 72 h only. In the hypothalamus, TRH levels was lowered after 3 and 24 h, and returned to the control after 72 h. Pilocarpine-induced seizures also elevated the TRH level after 72 h in the majority of the above structures, except for the hypothalamus and amygdala where no changes were found at any time point. A radioreceptor assay showed that kainate decreased the Bmax value of TRH receptors in the striatum and hippocampus after 3 and 24 h, respectively, and had no effect on the Kd values. In contrast, pilocarpine-induced seizures lowered the Bmax of TRH receptors in the striatum, hippocampus and piriform cortex after 72 h only, and decreased Kd values in the striatum, amygdala and frontal cortex. These data showed that pilocarpine- and kainate-induced seizures enhanced likewise preproTRH mRNA in the dentate gyrus; on the other hand, they differed with respect to time- and structure-related changes in TRH tissue levels and TRH receptors. These differences may have functional significance in TRH-dependent control mechanism of the seizure activity in these two models of limbic epilepsy.