THE UPTAKE OF γ-[3H]AMINOBUTYRIC ACID BY SLICES FROM VARIOUS REGIONS OF RAT BRAIN AND THE EFFECT OF LITHIUM

Abstract— Slices from various regions of rat brain, incubated at 25°C, rapidly accumulate [³H]GABA from the surrounding medium until after 60min tissue:medium ratios as high as 300 may be achieved. Kinetic analysis has demonstrated two distinct uptake systems for GABA in all the brain regions examined. One system has a relatively high substrate affinity ( K_m = 1.2 ± 10^-5 M) while the other has a lower affinity ( K_m = 4 ± 10^-4 M). Studies at low GABA concentration (5 ± 10^-8 M), as well as estimates of maximum velocities, have shown that the distribution of the high affinity uptake system is heterogeneous. Cortex, hypothala mus, midbrain and hippocampus have relatively high uptake rates while the striatum, cerebellum and pons and medulla have a lower uptake rate. Maximum velocities for the low affinity uptake system show much less regional variation.
Lithium, either added to the incubation medium or fed to rats, had no effect on the uptake of GABA by cortical slices.     

Pharmacological manipulation of gamma-aminobutyric acid (GABA) in morphine analgesia,tolerance and physical dependence

The findings from our laboratory indicated that pharmacological manipulations of GABA system modified morphine analgesia, tolerance and physical dependence. Elevating brain levels of GABA by slowing its destruction with aminooxyacetic acid not only antagonized the analgesic action of morphine in both non-tolerant and tolerant mice, but also enhanced the development of tolerance and physical dependence. On the other hand, blockade of postsynaptic sites of GABA receptors by bicuculline resulted in an inhibition of tolerance and dependence development. Administration of 2,4-diaminobutyric acid, an inhibitor of GABA uptake in the neurons, antagonized morphine analgesia in both non-tolerant and tolerant mice. However, it did not modify naloxone precipitated withdrawal jumping. On the contrary, β-alanine, an inhibitor of the GABA uptake process in glial cells, potentiated naloxone precipitated withdrawal jumping in morphine dependent mice, but it had no effect on morphine antinociception in both non-tolerant and tolerant mice.     

Ethanol Alters Kinetic Characteristics and Function of Striatal Morphine Receptors

Abstract: Morphine was shown to promote dopamine (DA) synthesis and release in mouse striatum, but mice rendered tolerant and dependent on ethanol were found to be less responsive to morphine's effects on striatal DA metabolism than control animals. Ethanol feeding also produced a change in the affinity of striatal "opiate" receptors for [³H]dihydromorphine, and these ethanol-induced receptor changes may be responsible for the altered biological effect of morphine.     

Effect of Chronic Administration of Morphine, U-50, 488H and [D-Pen, D-Pen]enkephalin on the Concentration of cGMP in Brain Regions and Spinal Cord of the Mouse

Bhargava, H. N. and Y. J. Cao. Effect of chronic administration of morphine, U-50,488H and [ -Pen², -Pen⁵]enkephalin on the concentration of cGMP in brain regions and spinal cord of the mouse. Peptides 18(10) 1629–1634, 1997.—The effects of chronic administration and subsequent withdrawal of μ-, κ- and δ-opioid receptor agonists on the levels of cyclic GMP in several brain regions and spinal cord of mice were determined in an attempt to further study the role of NO cascade in opioid actions. The agonists at μ-, κ- and δ-opioid receptor included morphine, U-50,488H and DPDPE, respectively. Tolerance to morphine was associated with highly significant increases in cGMP levels in corpus striatum (41%), cortex (36%), midbrain (73%) and cerebellum (51%) relative to controls. Abstinence caused increases in cGMP levels in corpus striatum (61%) and pons and medulla (45%). Tolerance to U-50,488H resulted in increases in cGMP levels in midbrain (52%) whereas abstinence from U-50,488H increased the cGMP levels in pons and medulla(76%). Tolerance to DPDPE was associated with increases in cGMP levels in hypothalamus (12%) and pons and medulla (33%) but decreases in cerebellum (66%) and spinal cord (58%). Abstinence from DPDPE produced increases in cGMP levels in pons and medulla (14%) but decreases in cerebellum (67%) and spinal cord (50%). Overall treatment with morphine and U-50,488H produced increases in cGMP levels in brain regions whereas DPDPE produced decreases in brain regions and spinal cord. Previous studies have shown that chronic administration of μ- and κ- opioid receptor agonists induce NO synthase (NOS) in certain brain regions and that the inhibitors of NO synthase attenuate tolerance to μ- and κ- but not to δ-opioid receptors agonists. Since activation of NO increases the production of cGMP, the present results demonstrating alterations of cGMP levels by μ-, κ- and δ-opioid receptor agonists are consistent with the behavioral results with NOS inhibitors on tolerance to μ-, κ- and δ-opioid receptor agonists.     

Opiate antagonist binding sites in discrete brain regions of spontaneously hypertensive and normotensive Wistar-Kyoto rats

The binding of 3H-naltrexone, an opiate receptor antagonist, to membranes of discrete brain regions and spinal cord of 10 week old spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats was determined. The brain regions examined were hypothalamus, amygdala, hippocampus, corpus striatum, pons and medulla, midbrain and cortex. 3H-Naltrexone bound to membranes of brain regions and spinal cord at a single high affinity site with an apparent dissociation constant value of 3 nM. The highest density of 3H-naltrexone binding sites were in hippocampus and lowest in the cerebral cortex. The receptor density (Bmax value) and apparent dissociation constant (Kd value) values of 3H-naltrexone to bind to opiate receptors on the membranes of amygdala, hippocampus, corpus striatum, pons and medulla, midbrain, cortex and spinal cord of WKY and SHR rats did not differ. The Bmax value of 3H-naltrexone binding to membranes of hypothalamus of SHR rats was 518% higher than WKY rats but the Kd values in the two strains did not differ. It is concluded that SHR rats have higher density of opiate receptors labeled with 3H-naltrexone in the hypothalamus only, in comparison with WKY rats, and that such a difference in the density of opiate receptors may be related to the elevated blood pressure in SHR rats.     

Opiate withdrawal induces dynamic expressions of AMPA receptors and its regulatory molecule CaMKIIalpha in hippocampal synapses

Adaptive changes in brain areas following drug withdrawal are believed to contribute to drug seeking and relapse. Cocaine withdrawal alters the expression of GluR1 and GluR2/3 subunits of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors in nucleus accumbens or amygdala, but the influence of drug withdrawal on hippocampus is little known. Here, we have examined the expression of GluR1 and GluR2/3 in hippocampal membrane and synaptic fractions following repeated morphine exposure and subsequent withdrawal. Repeated morphine exposure for 12 d increased GluR1 and GluR2/3 in synaptosome but not in membrane fraction. Interestingly, CaMKIIalpha, known to be able to regulate the function of AMPA receptors, was decreased in synaptosome but not in membrane fraction; pCaMKIIalpha, the phosphorylated form of CaMKIIalpha, was increased in both fractions. However, during opiate withdrawal, GluR1 was generally reduced while GluR2/3 was prominently increased in both fractions; pCaMKIIalpha was strongly decreased immediately after withdrawal, but detectably increased in late phase of morphine withdrawal in both fractions. Importantly, the opiate withdrawal-induced increase in GluR2/3 was dependent on the activation of glucocorticoid receptors and NMDA receptors, as it was prevented by the glucocorticoid receptor antagonist RU38486, or intrahippocampal injection of the NMDA receptor antagonist AP-5 or the antagonist to NR2B-containing NMDA receptors, Ro25-6981. These findings indicate that opiate withdrawal induces dynamic expression of GluR1 and GluR2/3 subunits of AMPA receptors in hippocampal synapses, possibly revealing an adaptive process of the hippocampal functions following opiate withdrawal.     

Modulation of opioid system in C57 mice after repeated treatment with morphine and naloxone: biochemical and behavioral correlates

C57 BL/6J (C57) mice display a particular pattern of responses following morphine administration, such as a rapid development of tolerance to the pharmacological action of the opiate and an increase in locomotor activity after a single injection of the drug. We have measured met-enkephalin content and the responsiveness of different opiate receptors after repeated administration of morphine and naloxone. Prolonged morphine administration changes neither met-enkephalin levels, nor the density of the opiate receptors in mice brain. In contrast repeated administration of the opiate antagonist naloxone, produced a marked increase in the number of 3H- DHM and 3H- DADLE binding sites in striatum and brainstem without modifying met-enkephalin concentrations. Behavioral studies have indicated that the morphine-induced increase in locomotor activity is enhanced in naloxone pretreated mice, thus suggesting a possible correlation between the behavioral response to morphine in C57 mice and the higher number of opiate receptors in the striatum.     

HIGH AFFINITY UPTAKE SYSTEMS FOR TAURINE IN TISSUE SLICES AND SYNAPTOSOMAL FRACTIONS PREPARED FROM VARIOUS REGIONS OF THE RAT CENTRAL NERVOUS SYSTEM. CORRECTION OF TRANSPORT DATA BY DIFFERENT EXPERIMENTAL PROCEDURES

Abstract— —The uptake of taurine into tissue slices of specific regions of the rat central nervous system (CNS) was compared with the uptake of taurine into synaptosomal fractions prepared from the corresponding regions. Two different techniques for performing control experiments were also compared: procedure I, correction for the uptake of taurine obtained from duplicate incubations but at 2°c and procedure II, correction of taurine uptake into extracellular or extrasynaptosomal space measured by inulin uptake experiments plus correction for diffusion (non-saturable) processes.
Kinetic analyses of the uptake data in tissue slices utilizing the procedure I correction technique indicate that six regions of the rat CNS (spinal cord, diencephalon, cortex, striatum, hippocampus, and midbrain) possess high affinity uptake systems (K_m values approx 60 μM or less). The K_m value for the cerebellum (105.4 ± 15.7 μM) is intermediate between a high and low affinity uptake system while the K_m value for the pons-medulla (210.0 12.4 μM) is considered to be low affinity. When procedure II techniques were utilized for correcting the uptake data all eight regions demonstrated high affinity uptake systems (11.8–73.2μM).
Synaptosomal fractions prepared from the spinal cord, pons-medulla, diencephalon, and midbrain demonstrate high affinity uptake systems (procedure I) for taurine (10.3–47.2 μM) while the hippocampus, cortex, striatum, and cerebellum have intermediate (but still high affinity) values (59.4–96.4 μM). High affinity uptake systems (8.2–79.8 μM) were obtained for all eight regions of the rat CNS when procedure II was utilized for correction of the data.     

Effect of acute and chronic cholinesterase inhibition with diisopropylfluorophosphate on muscarinic, dopamine, and GABA receptors of the rat striatum

The effects of acute and chronic administration of diisopropylfluorophosphate (DFP) to rats on acetylcholinesterase (AChE) activity (in striatum, medulla, diencephalon, cortex, and medulla) and muscarinic, dopamine (DA), and gamma-aminobutyric acid (GABA) receptor characteristics (in striatum) were investigated. After a single injection of (acute exposure to) DFP, striatal region was found to have the highest degree of AChE inhibition. After daily DFP injections (chronic treatment), all brain regions had the same degree of AChE inhibition, which remained at a steady level despite the regression of the DFP-induced cholinergic overactivity. Acute administration of DFP increased the number of DA and GABA receptors without affecting the muscarinic receptor characteristics. Whereas chronic administration of DFP for either 4 or 14 days reduced the number of muscarinic sites without affecting their affinity, the DFP treatment caused increase in the number of DA and GABA receptors only after 14 days of treatment; however, the increase was considerably lower than that observed after the acute treatment. The in vitro addition of DFP to striatal membranes did not affect DA, GABA, or muscarinic receptors. The results indicate an involvement of GABAergic and dopaminergic systems in the actions of DFP. It is suggested that the GABAergic and dopaminergic involvement may be a part of a compensatory inhibitory process to counteract the excessive cholinergic activity produced by DFP.     

Pyritinol inhibition of the GABA and benzodiazepine receptor

In 100 and 200 mumol/l concentration, pyritinol inhibited GABA binding to the GABA receptors of brain synaptosomal membranes. GABA receptors from the cerebral cortex, diencephalon and striatum were inhibited to approximately the same degree; those from the cerebellum and spinal cord were inhibited more. Both high and low affinity receptors were inhibited. Pyritinol did not greatly affect the number of binding sites (Bmax), but reduced the affinity (raised the dissociation constant KD) of both receptors. The benzodiazepine receptor, which is connected with the postsynaptic GABAA receptor, was also inhibited by pyritinol. The character of inhibition was the same as for GABA receptors, i.e. there was no change in the number of binding sites, but there was a decrease in their affinity. It is assumed that the similarity of the effect on GABA and benzodiazepine receptors is associated with their occurrence on one, or on two relatively firmly interconnected, protein molecules. Depression of the affinities of GABA and the associated benzodiazepine receptor, together with inhibition of GABA synthesis, in the presence of pyritinol indicate that diminished activity of the GABA system in the brain might be related to the activating effect of pyritinol.     

Is morphine dependence mediated exclusively by the Mu receptor?

Regional cerebral glucose utilization (RCGU) and behavior during naloxone precipitated withdrawal were studied in rats made dependent on either sufentanil, a highly selective mu opiate agonist, or morphine. Changes in RCGU during withdrawal in 23 of 24 anatomically related limbic and brainstem structures were indistinguishable between rats dependent on morphine or sufentanil. Linear regression analysis indicated that RCGU changes during withdrawal in these two groups were highly correlated (r=0.95). Withdrawal behaviors (autonomic signs of withdrawal, jumps, weight loss, and incidence of diarrhea) were likewise indistinguishable between the two groups. The correlations of both these measurements of dependence suggest that chronic occupation of mu opiate receptors alone is sufficient to produce dependence that is indistinguishable from morphine dependence.     

Role of corticotropin-releasing factor receptor-1 in opiate withdrawal

Previous studies indicate that corticotropin-releasing factor (CRF) contributes to the anxiety-like and aversive states associated with drug-induced withdrawal. The present study extends this work by analyzing the CRF receptor subtype involved in withdrawal responses. First, the influence of a selective CRF receptor-1 (CRF-R1) antagonist, CP-154,526, on opiate withdrawal behavior was examined. Pretreatment with the CRF-R1 antagonist significantly attenuated several behavioral signs of naltrexone-induced morphine withdrawal, including writhing, chewing, weight loss, lacrimation, salivation, and irritability, measured during the first hour of withdrawal. Next the expression of CRF-R1 was determined as a second measure of the involvement of this receptor in opiate withdrawal. Naltrexone-induced morphine withdrawal resulted in down-regulation of CRF-R1 mRNA in several brain regions, including the frontal cortex, parietal cortex, striatum, nucleus accumbens, and amygdala, but not in the hypothalamus or periaqueductal gray. Expression of CRF-R2, the other major CRF receptor subtype, was not down-regulated significantly by withdrawal in any of the regions examined, although morphine alone significantly increased levels of this receptor subtype. Taken together, the behavioral and receptor regulation findings indicate that CRF-R1 is the primary mediator of the actions of the CRF system on opiate withdrawal, although it is possible that CRF-R2 contributes to the response.     

Regional and Subcellular Calmodulin Content of Rat Brain

Calmodulin contents of cortex, cerebellum, striatum, diencephalon, and medulla + pons and of subcellular fractions of each region were determined by radioimmunoassay. The diencephalon had the highest level of calmodulin (48.87 +/- 4.56 micrograms/mg protein), whereas medulla + pons had the lowest level (8.01 +/- 0.84 micrograms/mg protein). In all brain regions, the mitochondrial fraction was richest in calmodulin (from 71 to 227 micrograms/mg protein) whereas other areas contained from 6 to 66 micrograms/mg protein.     

Effect of withdrawal of diazepam or morphine treatment on gastric motility (charcoal meal test) in mice: possible role of different central and peripheral receptors

Increased gastrointestinal motility in mice as one of the withdrawal symptoms of commonly abused drugs like diazepam or morphine and its possible mechanism of action was studied. Male Laka mice (20-25 g) were made addict to either diazepam (20 mg/kg, ip for 7 days) or morphine (10 mg/kg, sc for 9 days). Withdrawal symptoms were noted 24 hr after the last injection of diazepam or morphine. The animals were injected with Ro 15-1788 (flumazenil) (1 mg/kg, ip) or naloxone (2 mg/kg, ip) in the respective group to precipitate the withdrawal symptoms. Gastrointestinal motility was assessed by charcoal-meal test. Animals developed tolerance to acute sedative effect of diazepam, and similarly to the acute nociceptive action of morphine. On abrupt cessation of these drugs after chronic treatment the animals showed hyperlocomotion and hyperreactivity in diazepam withdrawal group and hyperalgesia on hot plate in morphine withdrawal groups, respectively. Increase in gastrointestinal motility was observed in all the drug withdrawal groups. Treatment with respective antagonists, Ro 15-1788 (flumazenil) and naloxone precipitated the withdrawal symptoms. The results suggest the involvement of both central and peripheral receptors of benzodiazepines and opioid (mu) receptors in the withdrawal symptoms of the benzodiazepines and morphine, respectively.     

The NMDA antagonist dizocilpine (MK801) attenuates motivational as well as somatic aspects of naloxone precipitated opioid withdrawal.

The non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist dizocilpine has recently been reported to antagonize certain overt withdrawal signs in morphine dependent rats. The purpose of the present study was to reassess this response and examine the effect of this drug in a model presumably reflective of the motivational impact of withdrawal using the place conditioning technique. Rats were made opiate dependent by the subcutaneous implantation of a 75 mg morphine pellet. Three-4 days later withdrawal was precipitated by naloxone 0.5 mg/kg. Dizocilpine (0.1-0.5 mg/kg) attenuated many of the subsequent behaviours elicited by naloxone, notably diarrhoea, mouth movements, paw shakes and ptosis. In a separate group of morphine dependent rats, naloxone (0.05 mg/kg) precipitated withdrawal produced a clear place aversion. This place aversion was blocked by dizocilpine (0.02-0.1 mg/kg) pre-treatment prior to conditioning. Therefore dizocilpine may modify both motivational and somatic aspects of opioid withdrawal.     

Effects of morphine administration and its withdrawal on rat brain aminopeptidase activities

The endogenous opioid neuropeptide system seems to be involved in the neural processes which underlie drug addiction. Several studies have reported that the administration of morphine induces changes in the levels and/or activity of endogenous opioid peptides (enkephalin, dynorphin) and their precursors in specific brain regions of the adult CNS. The aim of this work was to study the effects of chronic morphine exposure and its withdrawal on certain aminopeptidases capable of degrading opioid peptides in brain areas including the amygdala, hypothalamus, hippocampus, striatum and brain cortices. In animals treated with morphine, aminopeptidase N presented higher enzyme activity levels in the striatum, the hypothalamus and the amygdala compared to control animals, although statistically significant differences were observed only in the case of the striatum. In addition, the activity of soluble puromycin-sensitive aminopeptidase (PSA) was found to be higher in the frontal cortex of these rats. In contrast, rats experiencing withdrawal symptoms presented decreased levels of aminopeptidase activity in certain brain areas. Thus, the activity of aminopeptidase N in the hippocampus and soluble puromycin-sensitive aminopeptidase in the frontal cortex were found to be lower in rats experiencing naloxone precipitated withdrawal symptoms, compared to the corresponding controls. Finally, the activity of the three studied aminopeptidases in vitro was unaltered by incubation with morphine, suggesting that the observed effects are not due to a direct action of this opioid upon the aminopeptidases. The results of the present report indicate that aminopeptidases may play an important role in the processes of tolerance and withdrawal associated with morphine administration.     

Selective changes of receptor binding in brain regions of aged rats

Binding to several receptors was compared in brain regions of 3 and 21-23 month-old rats. In crude membrane preparations of aged rats the number of dopamine antagonist receptors in striatum was much reduced (-53%). beta-Noradrenergic receptors (cortex) and benzodiazepine receptors (hippocampus and cerebellum) were less but significantly reduced and serotonergic receptors, alpha 1 noradrenergic receptors (both in cortex) and dopamine agonist receptors (striatum) were unchanged. For each receptor binding the KD values were the same in young and old animals. GABA receptor binding (hippocampus and cerebellum) evaluated at only one 3H-GABA concentration (8 nM) was similar in both groups when expressed per protein content but significantly reduced in aged rats when expressed per tissue wet weight because of the partial purification of the synaptic membranes used for 3H-GABA binding. In our experimental conditions age-related changes of specific binding sites in the central nervous system were selective for some receptors studied and did not seem to be due to general non-specific modification of brain tissue composition.     

Long-term treatment with SR141716A, the CB1 receptor antagonist, influences morphine withdrawal syndrome

The role of the cannabinoid system in morphine withdrawal was examined through long-term CB1 receptor antagonist administration in morphine pellet implanted rats. SR141716A chronic treatment (5mg/kg i.p. twice a day for four days) did not influence the development of tolerance to the morphine analgesic effect but significantly reduced the intensity of naloxone-induced opiate withdrawal in tolerant rats: Specifically there was a significant reduction in the number of digging, teeth chattering and penile licking and the incidence of diarrhoea while other signs such as writhing, head dog shakes and rearing were unaffected. These results suggest that the pharmacological treatment with SR141716A could be of some interest in ameliorating opiate withdrawal syndrome.     

Benzodiazepine Treatment Causes Uncoupling of Recombinant GABAA Receptors Expressed in Stably Transfected Cells

Abstract: GABA_A and benzodiazepine receptors are allosterically coupled, and occupation of either receptor site increases the affinity of the other. Chronic exposure of primary neuronal cultures to benzodiazepine agonists reduces these allosteric interactions. Neurons express multiple GABA_A receptor subunits, and it has been suggested that uncoupling is due to changes in the subunit composition of the receptor. To determine if uncoupling could be observed with expression of defined subunits, mouse Ltk⁻ cells stably transfected with GABA_A receptors (bovine α1, β1, and γ2L subunits) were treated with flunitrazepam (Flu) or clonazepam. The increase in [³H]Flu binding affinity caused by GABA (GABA shift or coupling) was significantly reduced in cells treated chronically with the benzodiazepines, whereas the K _D and B _max of [³H]Flu binding were unaffected. The uncoupling caused by clonazepam treatment occurred rapidly with a t _1/2 of ∼30 min. The EC₅₀ for clonazepam treatment was ∼0.3 µ M , and cotreatment with the benzodiazepine antagonist Ro 15-1788 (5.6 µ M ) prevented the effect of clonazepam. The uncoupling observed in this system was not accompanied by receptor internalization, is unlikely to be due to changes in receptor subunit composition, and probably represents posttranslational changes. The rapid regulation of allosteric coupling by benzodiazepine treatment of the stably transfected cells should provide insights to the mechanisms of coupling between GABA_A and benzodiazepine receptors as well as benzodiazepine tolerance.