ALTERATIONS IN RECEPTORS CONTROLLING DOPAMINE SYNTHESIS AFTER CHRONIC ETHANOL INGESTION

The influence of acute and chronic ethanol treatment and withdrawal on regulation of dopamine synthesis in striatal and mesolimbic areas of mouse brain was evaluated. Tyrosine hydroxylase activity was estimated by measuring in vivo DOPA accumulation after inhibition of aromatic amino acid decarboxylase. Eight hours after a single (3 g/kg) dose of ethanol, DOPA synthesis was increased and pimozide, a dopamine receptor antagonist, stimulated DOPA synthesis to the same degree in ethanol-treated and control animals. On the other hand, 8 h after withdrawal of animals from chronic ethanol treatment, endogenous dopamine synthesis was the same in ethanol-withdrawn and control animals, but the stimulation of dopamine synthesis produced by low doses of pimozide or haloperidol was significantly less in the animals that had consumed ethanol. This effect was even more apparent at 24h after withdrawal; by 3 days after withdrawal the decreased response of ethanol-withdrawn animals to the administration of dopamine receptor blockers was no longer statistically significant. At all time points tested, high doses of pimozide or haloperidol stimulated DOPA synthesis equally in control and ethanol-withdrawn animals. Chronic ethanol treatment and withdrawal may alter the coupling between dopamine receptors which regulate dopamine synthesis and tyrosine hydroxylase.     

Effect of chronic haloperidol treatment on dopamine-induced inositol phosphate formation in rat brain slices

The effects of chronic haloperidol administration on the accumulation of inositol phosphates were examined in rat brain slices pre-labeled with [³H]myo-inositol and incubated with various dopaminergic drugs. Rats were treated with haloperidol-decanoate or its vehicle (sesame oil) for two, four or six weeks. Dopamine and the selective D₁ agonist, SKF38393, induced a significant increase in lithium-dependent accumulation of [³H]inositol monophosphate (IP₁) in the frontal cortex, hippocampus and striatum of vehicle-treated animals, while the selective D₂ agonist quinpirole did not show any effect on IP₁ accumulation. The actions of dopamine and SKF38393 were blocked by the D₁ antagonist, SCH23390, but not by the D₂ antagonist, spiperone, in all three brain regions. Haloperidol treatment did not affect basal phosphoinositide turnover in the three brain regions. Four or six weeks of haloperidol treatment significantly decreased dopamine-induced IP₁ accumulation in the striatum (by 30% and 25%, respectively), but not in the frontal cortex and the hippocampus. Four weeks of treatment with haloperidol significantly decreased IP₁ levels in the striatal slices when measured in the presence of quinpirole. However, the accumulation of IP₁ measured in the presence of SKF38393 was not significantly altered after haloperidol treatment. The loss of dopamine-sensitive IP accumulation was not observed in the presence of spiperone after haloperidol treatment. The number, but not the affinity, of [³H]sulpiride binding sites in the striatum was significantly increased (by 34–46%) after chronic haloperidol treatment. A timecourse study suggests that the inhibition by chronic haloperidol treatment of dopamine-induced phosphoinositide hydrolysis may involve an effect secondary to an increase in the number of dopamine D₂ receptors in the striatum.     

Dopamine-mediated behaviour and 3H-spiperone binding to striatal membranes in rats after nine months haloperidol administration

Rats were treated with haloperidol (1.5mg/kg/day) in their drinking water for 9 months, with or without a subsequent withdrawal period of 7–10 days. Compared with controls, spontaneous locomotion and apomorphine-induced stereotypy were reduced in rats maintained on haloperidol whereas both behaviours were increased after the withdrawal period. Maximum specific ³H-spiperone binding to striatal membrane preparations was increased (about 65%) in drug-treated rats with or without a withdrawal period. The dissociation constant for ³H-spiperone binding was significantly increased only in those rats maintained on haloperidol with no withdrawal period. The increase in maximum binding of ³H-spiperone was larger than that reported after less prolonged administration of neuroleptics. The size of the change should be taken into account in assessing the increased ligand binding reported in post-mortem brains of schizophrenics.     

Brain neurotransmitter receptors after long-term haloperidol: dopamine, acetylcholine, serotonin, alpha-noradrenergic and naloxone receptors.

Since long-term neuroleptic therapy is known to alter brain dopaminergic sensitivity, we tested the effects of chronic haloperidol administration (10 mg/kg/day for over 3 weeks) on the amount of the dopamine receptors (using ³H-apomorphine and ³H-haloperidol) in various regions of the rat brain. To test whether the changes in dopamine receptors were selectively produced, we also assayed acetylcholine receptors (with ³H-quinuclidinyl benzilate or ³H-QNB), alpha-noradrenergic receptors (with ³H-WB-4101), ³H-serotonin receptors and ³H-naloxone receptors.The specific binding of ³H-haloperidol increased significantly by 34% in the striatum and by 45% in the mesolimbic region after long-term haloperidol. The specific binding of ³H-apomorphine also increased significantly by 77% in the striatum and 55% in the mesolimbic area. Although there was a small significant increase of 20% in specific ³H-serotonin binding in the striatum, no such increment occurred in the hippocampus or the cerebral cortex. No significantly different binding occurred for the other ³H-ligands in these brain regions except for a 13% increase in alpha-noradrenergic binding in the cerebral cortex. These results indicate that long-term haloperidol treatment produces rather selective increases in dopamine/neuroleptic receptors, without much change in 4 other types of receptors. Such relatively selective increments in these receptors may be the basis of dopaminergic supersensitivity (e.g. tardive dyskinesia) after long-term haloperidol.     

[3H]2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalen (ADTN)

The regional distribution and in vivo binding of the dopamine analog 2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalen (ADTN) was studied in the brain. The highest density of binding sites was in the striatum, with virtually no binding in the cerebellum. The binding of [³H]ADTN reflects an occupation of specific dopamine sites because the binding was diminished by the simultaneous administration of the dopamine antagonist haloperidol or the dopamine precursorl-3,4-dihydroxyphenylalanine (l-dopa). Chronic administration of haloperidol orl-dopa prior to assaying for in vivo binding resulted in an increase in the number of sites for [³H]ADTN which correlates to the increase observed in in vitro assays following long-term treatment with these agents. The subcellular distribution of in vivo labeled ADTN sites in the caudate nucleus indicate a high density of specific binding sites in the microsomal fraction, P₃. Overall, these data demonstrate that the aminotetralins, such as ADTN, which bind with high affinity to the dopamine receptor in the caudate nucleus in vitro and in vivo, can provide precise information on the topography of this receptor.     

Sub-chronic iron overload triggers oxidative stress development in rat brain: implications for cell protection

This work was aimed to test the hypothesis that sub-chronic administration of iron-dextran (Fe-dextran) (six doses of 50 mg Fe-dextran/kg) to rats triggers a transient oxidative stress in brain and mechanisms of cellular antioxidant defence. After 2 h of administration of the 6th dose, a significant increase of total Fe, the labile Fe pool (LIP), the lipid radical (LR^?)/α-tocopherol (α-T) content ratio were observed, as compared to values in control brain homogenates. The ascorbyl radical (A^?)/ascorbate (AH^?) content ratio and the oxidation rate of 2′,7′-dichlorodihidrofluorescein (DCFH-DA) were significantly higher in Fe-dextran treated rats, as compared to values in brain from control rats after 4 h treatment. An increase in both catalase (CAT) and superoxide dismutase (SOD) activity was observed at 8 and 1–2 h, respectively. No significant changes were detected in the nuclear factor-κB (NF-κB) levels in nuclear extracts from rat brains after 1–8 h of Fe-dextran administration. After 2 h of Fe administration Fe concentration in cortex, striatum and hippocampus was significantly increased as compared to the same areas from control animals. Both, CAT and SOD activities were significantly increased in cortex after Fe administration over control values, without changes in striatum and hippocampus. Taken as a whole, sub-chronic Fe administration enhances the steady state concentration of Fe in the brain LIP that favors the settlement of an initial oxidative stress condition, both at hydrophilic and lipophilic compartments, resulting in cellular protection evidenced by antioxidant enzyme upregulation.     

Neurochemical Changes in Brain Induced by Chronic Morphine Treatment: NMR Studies in Thalamus and Somatosensory Cortex of Rats

To investigate the effects of chronic morphine treatment and its cessation on thalamus and the somatosensory cortex, an ex vivo high resolution (500 MHz) ¹H nuclear magnetic resonance spectroscopy (NMRS), in the present study, was applied to detect multiple alterations of neurochemicals and/or neurometabolites in the rats. Ten days of chronic morphine administration was observed to markedly increase the total amount of lactate (Lac), myo-inositol (my-Ins) (each P < 0.01) and aspartate (Asp) (P < 0.05), and significantly decrease that of glutamate (Glu) and glutamine (Gln) in the rats thalamus (each P < 0.05). In the somatosensory cortex, chronic morphine was shown to increase the level of Lac and my-Ins, and decrease that of Glu (each P < 0.05). Interestingly, the ratio of Glu/GABA was found to decrease in these two brain areas after chronic morphine treatment, and among the detectable neurochemicals in those two cerebral areas, only taurine (Tau) showed to result in a significant increment in thalamus during the process of morphine discontinuation (P < 0.05). Moreover, the alterations of multiple neurochemicals due to chronic morphine exhibited a tendency of recovery to the normal level over the course of morphine withdrawal. The results suggested that, in thalamus and the somatosensory cortex, chronic morphine administration and its cessation could induce multiple neurochemical changes, which may involve in the brain energy metabolism, activity and transition of neurotransmitters.     

Effects of Typical and Atypical Antipsychotic Drugs on Rat Brain Muscarinic Receptors

Quantitative in vitro autoradiography was used to examine changes in muscarinic M1/M4 and M2/M4 receptors (targeted with [³H]pirenzepine and [³H]AF-DX384 respectively), in rats treated with the typical (haloperidol) and atypical (clozapine and olanzapine) antipsychotic medications for a period of 36 days. Rats were sacrificed at either 2 h or 48 h after the last drug administration to examine immediate effects as well as the effects at 48 h after drug withdrawal. Haloperidol significantly increased [³H]pirenzepine binding in the dentate gyrus (37%) and in the CA1 region of the hippocampus (34%) in animals sacrificed 2 h after the last drug administration compared to controls. Similarly, clozapine significantly increased [³H]pirenzepine binding in dentate gyrus (29%) in rats sacrificed 2 h after the last drug administration compared to controls. Haloperidol decreased [³H]AF-DX384 binding in the basolateral nucleus of the amygdala (20%) in the rats sacrificed 48 h after the last drug administration compared to controls. These findings suggest that muscarinic receptors and limbic brain regions such as hippocampus and amygdala might represent common targets that mediate beneficial clinical effects of antipsychotic drugs.     

Effects of chronic caffeine on brain adenosine receptors: Regional and ontogenetic studies

The effect of chronic caffeine treatment on three different binding sites in five brain areas of mice is characterized. The sites studied were the adenosine receptor, using [³H] diethylphenylxanthine, the benzodiazepine receptor, using [³H] diazepam and the adenosine uptake site, using [³H] nitrobenzylthioinosine. Significant increases were only observed in adenosine receptors with the greatest degree of change seen in the cerebellum and brain stem at both 16 and 23 days of caffeine treatment. The lack of significant effects of chronic caffeine on benzodiazepine receptors and adenosine uptake sites indicates that the caffeine effect is specific. The effect of chronic caffeine treatment on the ontogency of adenosine receptors was also studied with the result showing a significantly accelerated development of the receptor in the caffeine treated animals. The adult adenosine receptor levels were 20–30% higher than those observed in control animals. The observed alterations in adenosine receptor number which occur as a consequence of caffeine consumption may underlie some of the behavioral effects of this cortical stimulant as well as provide insights concerning the mechanisms of tolerance to and dependence on caffeine.     

Brain levels and turnover rates of presumptive neurotransmitters as influenced by administration and withdrawal of ethanol in mice

—Effects of acute or chronic administration of ethanol and its withdrawl on the steady-state levels and turnover rates of certain neurotransmitters have been investigated in mice. The influence of long-term administration of ethanol on the activities of enzymes involved in the metabolism of these transmitters has also been studied. Acute administration of ethanol or acetaldehyde or chronic administration of ethanol resulted in a decrease in the cerebral contents of acetylcholine, acetylCoA and CoA. Brain levels of 5-hydroxytryptamine, norepinephrine and choline remained unchanged after acute administration of ethanol. However, chronic administration of ethanol resulted in a decrease in the norepinephrine content without significantly affecting 5-hydroxytryptamine or choline contents. Cerebral levels of γ-aminobutyric acid increased with both acute or chronic administration of ethanol. The total incorporation of [³H]choline into acetylcholine in brain was depressed upon acute administration of ethanol. After withdrawal of ethanol for one day cerebral levels of norepinephrine returned to normal; however, γ-aminobutyric acid and acetylcholine returned to normal levels at 2 and 4 days after ethanol withdrawal, respectively. Pretreatment of mice with pyrazole, an inhibitor of alcohol dehydrogenase, prevented the ethanol-induced decrease in cerebral acetylcholine levels. The activities of cerebral choline acetyltransferase and glutamic decarboxylase were decreased after 2 weeks of chronic ethanol administration. However, the activities of acetyl cholinesterase and GABA-transaminase remained unaffected after 2 weeks of ethanol treatment     

The effect of chronic L-DOPA administration on supersensitive pre- and postsynaptic dopaminergic receptors in rat brain

Chronic administration of haloperidol induced supersensitivity of the pre- and postsynaptic dopaminergic receptors in rat brain. The response of the presynaptic receptors was determined by an enhanced inhibitory effect of apomorphine on dopamine synthesis after gamma-butyrolactone injection. This change in the receptor function was detected both in the nigrostriatal and mesolimbic pathways. Haloperidol also increased the ³H-spiperone binding sites in striatal membranes, indicating supersensitivity of the postsynaptic receptors. Subsequent prolonged treatment with high doses of L-DOPA/carbidopa resulted in a decrease in ³H-spiperone binding sites, but had no effect on the supersensitive presynaptic receptors. It is suggested that tardive dyskinesia may be a state of both pre- and postsynaptic dopamine receptor supersensitivity and that chronic L-DOPA treatment may have a differential effect on these sites.     

Protein synthesis in mitochondrial and microsomal fractions from rat brain and liver after acute or chronic ethanol administration

Incorporation of C¹⁴ Leucine was determined $\text{in vitro}$ or $\text{in vivo}$ in isolated mitochondria and microsomes of rat brain and liver after acute or chronic ethanol administration $\text{in vivo}$.The protein synthesis in mitochondrial and microsomal preparation was inhibited respectively by chloramphenicol and cycloeximide, specific inhibitors for the two systems tested. The experimental data demonstrate that the $\text{in vitro}$ protein synthesis in both systems, mitochondrial and microsomal, is strongly affected only after chronic treatment which produces significant activation at the mitochondrial and microsomal level in the liver and an inhibition on the same systems of the brain.The data for $\text{in vivo}$ protein synthesis instead show strong inhibition after acute administration, except for brain mitochondria, which are practically unaffected, while after chronic treatment no significant alterations are observed.     

Axonal transport in nigro-neostriatal neurons during morphine tolerance development and abstinence in rats.

Axonal transport of [³H]protein in the nigro-neostriatal pathway in rats was examined during acute and chronic morphine administration and during morphine abstinence. Two days after a microinjection of [³H]lysine into the left substantia nigra zona compacta, more than 95% of the radioactivity present in the rat forebrain was protein-bound. Examination of frozen frontal brain sections revealed that 80–90% of the labelled protein of the injected side was located in brain areas traversed by the nigro-neostriatal pathway. As a positive control, intranigrally administered colchicine reduced the amount of [³H]protein transported after 5 days to the nucleus caudatus-putamen (neostriatum) to approx 18-26% of control. In animals rendered morphine-dependent by subcutaneous implantation of tablets containing 75 mg of morphine base, 27–86% more radioactivity accumulated in the neostriatum at 3, 4 and 5 days after [³H]lysine injection. In contrast, 23–48% less radioactivity was recovered in the neostriatal areas of animals withdrawing from morphine 24 h after [³H]lysine. Gel electrophoresis of soluble and particulate [³H]protein fractions from neostriatal tissues indicated that the gel patterns of radioactivity were not altered by chronic morphine administration. Neither morphine administration nor morphine abstinence altered the rate or amount of [³H]lysine incorporation into protein of the substantia nigra. These data demonstrate that chronic morphine administration was accompanied by a generalized increase in the amount of labelled protein transported to the neostriatum but the procedure was not sufficiently sensitive to detect a minor qualitative alteration of any particular protein(s). Furthermore, these data suggest that either the capacity or the rate of nigro-neostriatal protein transport may be increased during chronic morphine administration in the rat.     

Assessment of sigma-1 receptor occupancy in mice with non-radiolabelled FTC-146 as a tracer

Abstract

The use of liquid chromatography coupled with mass spectrometry (LC-MS/MS) is advantageous in in-vivo receptor occupancy assays at pre-clinical drug developmental stages. Relatively, its application is effective in terms of high throughput, data reproducibility, sensitivity, and sample processing. In this perspective, we have evaluated the use of FTC-146 as a non-radiolabelled tracer to determine the sigma-1 receptor occupancy of test drugs in mice brain. Further, the brain and plasma exposures of test drug were determined at their corresponding occupancies. In this occupancy method, the optimized tracer treatment (sacrification) time after intravenous administration was 30?min. The tracer dose was 3?µg/kg and specific brain regions of interest were frontal cortex, pons and midbrain. Mice were pretreated orally with SA4503, fluspidine, haloperidol, and donepezil followed by tracer treatment. Among the test drugs, SA4503 was used as positive control group at its highest test dose (7?mg/kg, intraperitoneal). There was a dose-dependent decrease in brain regional FTC-146 binding in pretreated mice. From the occupancy curves of SA4503, fluspidine, haloperidol, and donepezil the effective dose (ED₅₀) value ranges are 0.74–1.45, 0.09–0.11, 0.11–0.12, and 0.07–0.09?mg/kg, respectively. Their corresponding brain effective concentration (EC₅₀) values are 74.3–132.5, 3.4–3.7, 122.5–139.5, and 8.8–11.0?ng/g and plasma EC₅₀ values are 34.3–53.7, 0.08–0.10, 7.8–9.5, and 0.6–0.7?ng/mL. Brain regional distribution and binding inhibition upon pretreatment were comparable with data reported with labeled [¹⁸F]FTC-146. Drug exposures were simultaneously determined and correlated with sigma-1 occupancy from the same experiment. Wide category drugs can be assayed for sigma-1 receptor engagement and their correlation with exposures aid in clinical development.     

Effects of alloxan-induced diabetes on somatostatin binding to cytosolic components of rabbit gastric fundic mucosa

Diabetes was induced by administration of alloxan (150 mg/Kg) to 24 h-fasted rabbits. Somatostatinlike immunoreactivity (SLI) and cytosolic binding sites for somatostatin in gastric fundic mucosa were studied using radiolabelled Tyr¹¹-somatostatin. Three months after the onset of the disease, the specific binding of somatostatin to these sites was seen to be significantly lower, due to a reduction in the number (but not the affinity) of specific somatostatin binding sites of high-affinity and a disappearance of the specific, somatostatin binding sites of low-affinity. These changes were associated with an increase in the SLI concentration in both gastric fundic mucosa and plasma.     

Brain γ-Aminobutyric Acid Turnover Rates After Spontaneous Chronic Ethanol Intake and Withdrawal in Discrete Brain Areas of C57 Mice

The effect of 4 weeks of spontaneous chronic ethanol intake in drinking water and then ethanol withdrawal on the gamma-aminobutyric acid (GABA) steady-state levels and turnover rates was investigated in 15 brain areas of C57 Bl/6J alcohol-preferring mice. These mice did not display typical ethanol withdrawal convulsions. There was no statistically significant difference in the brain GABA steady-state levels among the control group, chronic ethanol-treated mice, and mice after ethanol withdrawal. In contrast, chronic ethanol treatment induced significant variations in GABA turnover rate, as measured by gabaculine-induced accumulation of GABA, in eight of 15 areas examined versus a decrease in seven brain areas [cerebellum (-29%), amygdala (-28%), olfactory tubercles (-24%), septum (-24%), striatum (-53%), frontal cortex (-21%), and hippocampus (-24%)]; an increase in turnover rate in the posterior colliculus (100%) was observed. At 26 h after ethanol withdrawal, in the seven areas in which GABA turnover rate decreased after spontaneous chronic ethanol intake, a return to the initial control value was observed; in the posterior colliculus, the turnover rate did not change, remaining higher than the control value. This persisting alteration of GABA turnover rate may be related to the absence of the ethanol withdrawal syndrome in the C57 mouse strain.     

Polyamine turnover in different regions of rat brain

The dynamics of the formation and disappearance of polyamines in rat brain have been examined after intraventricular administration of a tracer dose of [³H]putrescine. After 2 days [³H]putrescine was no longer detectable in any brain region examined. [³H] Spermidine and [³H] spermine were formed in all brain areas. In the midbrain, hypothalamus and cerebellum (regions which manifested the greatest initial accumulation of tritium) the specific radioactivity of spermidine declined with a half-life of 16-19 days. However, in areas with a low initial accumulation of tritium (the medulla-pons, internal capsule, cerebral cortex and corpus striatum) the specific radioactivity of spermidine changed very little between 2 and 19 days after the putrescine administration. Levels of [³H]spermine increased continuously in all brain areas for a 14-day period after the putrescine injection.     

Comparative Effects of Chronic Haloperidol and Sulpiride Treatment on Nigral and Striatal GABA Content

Nigral and striatal GABA contents were assayed in male rats treated chronically with haloperidol or sulpiride, two dopamine-receptor blocking agents that have different neuropharmacological spectra in regard to their biochemical, behavioural, and clinical properties. No great difference was observed between the chronic effects of haloperidol and sulpiride on nigral and striatal GABA content. However, low doses (30 μg/kg, intraperitoneally) of the dopamine-receptor agonist apomorphine, injected 12 h after the discontinuation of chronic haloperidol or chronic sulpiride treatment, induced opposite changes in nigral GABA levels suggesting the existence of a different “status” of the dopamine receptors during the 12 h-period following the withdrawal of haloperidol or sulpiride.     

Enhanced striatal 3H-spiroperidol binding induced by chronic haloperidol treatment inhibited by peptides administered during the withdrawal phase

Chronic intragastric administration of haloperidol (1.5 mg/kg/day) for 21 days followed by a 3-day withdrawal period resulted in the development of enhanced locomotor activity response to apomorphine, and an increase in the number of binding sites for 3H-spiroperidol in the striatal membranes of the rat brain. Subcutaneous administration of Pro-Leu-Gly-NH2 or cyclo(Leu-Gly) in doses of 2 mg/kg/day given for 3-days after termination of haloperidol treatment inhibited the enhanced response to apomorphine, as well as the increases in the number of 3H-spiroperidol binding sites in the striatum. If indeed, the supersensitivity of striatal dopamine receptors is one of the mechanisms in the development of tardive dyskinesia symptoms, the present results suggest that the above peptides may be helpful in ameliorating some of the symptoms of tardive dyskinesia induced by neuroleptic drugs.     

Reduction in the number of serotonin receptors in the brainstem of morphine dependent rats: Relation to blockade of naloxone precipitated jumping by serotonin agonists

The effect of various drugs was studied on the naloxone-precipitated withdrawal syndrome of morphine-dependent rats. Quipazine and metachlorophenylpiperazine, two agents mimicking serotonin at central receptors, markedly lowered the frequency of jumping in abstinent rats; m-chlorophenylpiperazine significantly blocked diarrhea as well. Ptosis and diarrhea but not jumping were significantly reduced by clonidine. None of the withdrawal signs considered was significantly affected by propranolol or haloperidol. A significant reduction in the number of ³H-serotonin binding sites was found in the brainstem of morphine-dependent rats, but no change was observed in the number of serotonin receptors in the cortex. The hypothesis is proposed that in morphine dependent rats a central serotoninergic hypofunction, probably related to a decrease in the number of serotonin receptors in the brainstem, occurs as a consequence of persistent activation of central serotonin function by long-term treatment with morphine. This mechanism appears to play a major role in the compulsive jumping shown by morphine-dependent rats after naloxone injection. Alpha₂ adrenergic sites may play a role in the manifestation of other withdrawal signs examined in this study.