Regional distribution of low-affinity GABA receptors coupled to benzodiazepine receptor subtypes in rat brain: an autoradiographic evaluation

Quantitative autoradiography of rat brain coronal sections show that maximum enhancement (more than 80%) of [3H]flunitrazepam binding by GABA occurs in brain regions particularly rich in type I benzodiazepine receptors (inferior colliculus, medium raphe, central gray and substantia nigra); conversely, brain areas where type II predominates show the lowest enhancement by GABA (about 50%). These results, suggesting that the coupling of GABA receptors with type I sites is more efficient than that with type II sites, are at variance with those reported on GABA-benzodiazepine receptors expressed in transfected cells, where the greater GABA potentiation of benzodiazepine binding is due to a subtype of the type II site containing the alpha 3 subunit of the GABAA receptor. One possible explanation of these discrepancies is that the type II receptors found in type I-enriched tissues (inferior colliculus, median raphe, central gray and substantia nigra) are associated with the alpha 3-subunit, while the type II sites present in limbic and cortical regions represent a subpopulation carrying the alpha 2-subunit of the GABAA receptor, with lower GABA potentiation.     

Effect of acute and chronic ethanol administration on serotonin turnover in rat brain

In rats treated acutely and chronically with ethanol, brain serotonin (5-HT) turnover was determined by measuring in the same animals the rate of accumulation of 5-HT and the rate of decline of 5-hydroxyindoleacetic acid (5-HIAA) in the brain after administration of the MAO inhibitor pargyline-HCl. In the acute study, 5-HT turnover was not altered after a single 4 g/kg oral dose of ethanol. In two separate studies, it was shown that chronic administration of ethanol in the form of a liquid diet for 32 days also did not alter brain 5-HT turnover, even when the rats had developed a significant degree of tolerance to the motor-impairing effects of ethanol. These results suggest that tolerance to ethanol is unrelated to any change in brain 5-HT turnover.     

Autoradiographic studies on benzodiazepine receptor subtypes in the rat brain

The technique of in vitro autoradiography which was developed by Kuhar and others was applied to the rat brain with use of 3H-flunitrazepam (flu) as a radioactive ligand. The cerebral cortex, hippocampus, substantia nigra and cerebellar cortex were rich in 3H-flu binding sites. To differentiate the benzodiazepine receptor (BZR) subtype, the authors used a type 1 specific ligand, either triazolopiridazine (CI 218872) or methyl-beta-carboline-carboxylate (beta-CCM), as an unlabeled displacer. The preparations were exposed on a 3H-sensitive film and then the film was developed. Computer-analysis of thus obtained autoradiographic pictures revealed that type 2 binding sites were distributed evenly within the rat brain, but with slight predominance in the hippocampus. After adding beta-CCM, no silver grains were noticed in the cerebellum and substantia nigra. These data meant that these two structures contained essentially type 1 BZR, while the hippocampus contained both type 1 and type 2 receptors. Autoradiographically, characteristic distribution of BZR represented by 3H-flu binding was considerably lost by adding a type 1 specific ligand, and this treatment caused the silver grains to be evenly distributed. These data suggest that the BZR which is directly associated with characteristic pharmacological actions such as anxiolytic and hypnotic effects is type 1, and type 2 binding sites have a less characteristic distribution pattern and might be pharmacologically less specific.     

Effect of acute and chronic pentylenetetrazol treatment on benzodiazepine and cholinergic receptor binding in rat brain

The binding of [3H] diazepam and [3H] flunitrazepam in rat cerebral cortex was not altered by either acute or chronic administration of pentylenetetrazol except in rats made to convulse 30 min before sacrifice. Rats treated for up to 6 months with doses of pentylenetetrazol which are below seizure threshold in naive rats, became increasingly sensitive to the CNS stimulant effect of pentylenetetrazol as demonstrated by the development of myoclonus and convulsions during treatment periods. These effects were not correlated with any changes in benzodiazepine binding in cerebral cortex or cerebellum and [3H] quinuclidinyl benzilate binding in cerebral cortex. Acute convulsant doses of pentylenetetrazol increased benzodiazepine binding in cerebral cortex, but only in those rats which actively convulsed. Benzodiazepine and cholinergic receptors of the cortex, and benzodiazepine receptors of the cerebellum, therefore, do not appear to change with either the acute or chronic subconvulsive administration of pentylenetetrazol.     

Effect of ethanol administration and withdrawal on benzodiazepine receptor binding in the rat brain

Administration of single doses of ethanol or presence of ethanol in the incubation medium does not change 3H-flunitrazepam binding in rat cerebral cortex, hippocampus and cerebellum. In rats made dependent on ethanol by three daily ethanol administrations for six days and sacrifices 1 hr after the last ethanol dose 3H-flunitrazepam binding is decreased in the cerebral cortex and cerebellum. This decrease is present only in membranes preincubated with Triton X-100 and is due to decreased number of binding sites. In rats sacrificed 16 hours after the last chronic ethanol dose the 3H-flunitrazepam binding returns to control levels.     

Effect of chronic estradiol treatment on brain dopamine receptor reappearance after irreversible blockade: an autoradiographic study.

Quantitative autoradiography was used to investigate dopamine receptor repopulation kinetics after irreversible dopamine receptor inactivation with N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ). The striatum and substantia nigra of two groups of ovariectomized female rats were compared. One group of rats was pretreated with estradiol (10 micrograms, twice daily, for 2 weeks), and another group received the vehicle. Striatal D1 dopamine receptors had larger degradation and production rate constants, compared with D2 receptors. The D2 receptor degradation rate constant increased rostro-caudally in the striatum of vehicle-treated rats, whereas this was not observed for estradiol-treated animals. A trend similar to that for D2 receptors was observed for the D1 receptor degradation rate constant in the striatum of vehicle-treated rats, whereas in estradiol-treated animals this constant decreased rostro-caudally. In the anterior and the middle parts of the striatum D2 receptor recovery parameters were not affected by chronic estradiol treatment, but in the posterior part estradiol-treated rats had lower receptor degradation and production rate constants. In the anterior part of the striatum, chronic estradiol treatment did not affect the recovery parameters of D1 receptors, whereas lowered receptor degradation and production rate constants were observed in the middle and posterior parts. D1 receptor recovery parameters in the substantia nigra were not affected by chronic estradiol treatment. After EEDQ administration to vehicle-treated rats, striatal dopamine levels decreased gradually, to reach a minimum 4 days later, and returned to control values after 7 days. In estradiol-treated rats, however, dopamine levels increased 2 days after EEDQ. Levels of the dopamine metabolites dihydroxyphenylacetic acid and homovanillic acid increased in the striatum after EEDQ administration in vehicle-treated rats. Even greater increases that lasted longer were observed in estradiol-treated rats after EEDQ. Striatal levels of serotonin and its metabolite 5-hydroxyindoleacetic acid were not significantly affected by EEDQ or estradiol administration. In summary, estradiol decreased striatal D1 and D2 receptor degradation rate constants, with the greatest effect being observed in the caudal part of the striatum. EEDQ dopamine receptor inactivation also revealed an increase of dopamine and its metabolites in the striatum after estradiol treatment.     

Effects of acute and long-term administration of escitalopram and citalopram on serotonin neurotransmission: an in vivo electrophysiological study in rat brain.

The present study was undertaken to compare the acute and long-term effects of escitalopram and citalopram on rat brain 5-HT neurotransmission, using electrophysiological techniques. In hippocampus, after 2 weeks of treatment with escitalopram (10 mg/kg/day, s.c.) or citalopram (20 mg/kg/day, s.c.), the administration of the selective 5-HT(1A) receptor antagonist WAY-100,635 (20-100 microg/kg, i.v.) dose-dependently induced a similar increase in the firing activity of dorsal hippocampus CA(3) pyramidal neurons, thus revealing direct functional evidence of an enhanced tonic activation of postsynaptic 5-HT(1A) receptors. In dorsal raphe nucleus, escitalopram was four times more potent than citalopram in suppressing the firing activity of presumed 5-HT neurons (ED(50)=58 and 254 mug/kg, i.v., respectively). Interestingly, the suppressant effect of escitalopram (100 microg/kg, i.v.) was significantly prevented, but not reversed by R-citalopram (250 microg/kg, i.v.). Sustained administration of escitalopram and citalopram significantly decreased the spontaneous firing activity of presumed 5-HT neurons. This firing activity returned to control rate after 2 weeks in rats treated with escitalopram, but only after 3 weeks using citalopram, and was associated with a desensitization of somatodendritic 5-HT(1A) autoreceptors. These results suggest that the time course of the gradual return of presumed 5-HT neuronal firing activity, which was reported to account for the delayed effect of SSRI on 5-HT transmission, is congruent with the earlier onset of action of escitalopram vs citalopram in validated animal models of depression and anxiety.     

Regulation of muscarinic receptor subtypes and their responsiveness in rat brain following chronic atropine administration

Chronic administration of l-atropine to rats caused a dose-dependent increase (30%) in the density of muscarinic receptors, as measured with [3H]quinuclidinyl benzilate ([3H]QNB], in cortex (CTX), dorsal hippocampus (DH), and heart but not the corpus striatum. Serum concentrations of l-atropine reached 80 to 160 nM within 6 hr, whereas densities of binding sites for [3H]QNB did not show a significant increase until after the second day of infusion and receptor densities did not reach new steady state levels until after the fourth day of infusion. The density of binding sites for the M1-selective muscarinic receptor antagonist, [3H]pirenzepine ([3H]PZ) was also measured. As noted previously, the density of binding sites for [3H]PZ (defined as M1) was lower than the density of binding sites for [3H]QNB (defined as M1 plus M2). When the densities of binding sites for [3H]QNB and [3H]PZ in CTX plus DH were determined after 14 days of treatment, [3H]QNB binding sites showed a 28% increase, whereas [3H]PZ binding sites did not show any increase. The difference between the densities of binding sites for [3H]QNB and [3H]PZ, an estimate of the density of M2 sites, doubled. The density of binding sites for [3H]QNB appeared to be stable for at least 64 hr after the withdrawal of the drug. The increase in the density of binding sites for [3H]QNB was not reflected in the binding of [3H]oxotremorine-M ([3H]OXO-M), a muscarinic agonist, which was unchanged by l-atropine administration. Because the binding of [3H]OXO-M is sensitive to GTP, this observation suggests that the "induced" receptors may not be coupled to a guanine nucleotide-binding protein. In spite of the fact that there was a doubling of the density of M2 sites, no significant differences in dose-response curves for carbachol-induced inhibition of [3H]cAMP accumulation were observed in slices of CTX plus DH from control and l-atropine-treated rats. Similarity, acetylcholine-stimulated accumulation of [3H]inositol phosphates in slices of CTX plus DH showed no significant differences between the tissues from control and treated rats.(ABSTRACT TRUNCATED AT 400 WORDS)     

Modification of serotonin metabolism in rat brain after acute or chronic administration of morphine

The steady state concentration of 5-hydroxyindoleacetic acid (5-HIAA) was elevated in rat brain for at least 4 hr after administration of a single dose of morphine sulfate (30 mg/kg, s.c.), and for more than 40 hr after subcutaneous implantation of pellets of morphine alkaloid. The concentration of 5-HIAA returned to normal 3 days after pellet implantation at a rate that paralleled the development of tolerance to the analgesic and other overt actions of morphine. Morphine did not modify the steady state concentration of serotonin under any of the treatment conditions. The turnover of brain serotonin was increased significantly during the 90-min period following a single injection of morphine sulfate (30 mg/kg, s.c.), as indicated by an increased rate of accumulation of 5-HIAA after blockade of efflux of 5-HIAA by probenecid in morphinetreated animals. As judged either by the rate of accumulation of 5-HIAA after administration of probenecid, or by the rate of accumulation of serotonin after treatment with pargyline, an increase in turnover of serotonin was evident in brains of tolerant rats 72 hr after pellet implantation. The rate of efflux of 5-HIAA from the brain was the same in control and morphine-tolerant rats. These results indicate that changes in brain serotonin metabolism are associated with both the acute effects of morphine and with morphine tolerance.     

Effect of acute and chronic anticonvulsant administration on endogenous γ-hydroxybutyrate in rat brain

The effect of acute and chronic administration of ethosuximide, trimethadione, sodium valproate, clonazepam, phenobarbital, and diazepam on brain concentrations of γ-hydroxybutyrate (GHB) was determined by gas-liquid chromatography. The dose and time to sacrifice for each drug was determined by testing for effectiveness against GHB-induced absence seizures in the rat using an automated frequency analysis for quantitation of the electrocorticogram. Acute administration of ethosuximide, trimethadione, and sodium valproate, produced an increase in whole brain GHB. Ethosuximide, trimethadione, and phenobarbital given chronically produced a decrease in whole brain GHB. All changes took place in subcortex and cerebellum. Acute ethosuximide treatment produced a greater increase in GHB concentration at higher doses. The acute changes with the drugs coincided with the onset of anticonvulsant effect, but were short-lived and, in the case of ethosuximide and trimethadione, followed by a significant depression in GHB concentrations. The anti-petit mal action of these anticon-vulsants may be related to their effect on GHB in brain.     

Affinity changes in muscarinic acetylcholine receptors in the rat brain following acute immobilization stress: an autoradiographic study.

The modifications in rat brain muscarinic acetylcholine receptors induced by acute immobilization stress lasting 10 min or 2 h were analyzed by quantitative in vitro autoradiography. [3H]N-Methylscopolamine ([3H]NMS) was used as a ligand. Immobilization stress for 10 min did not produce any significant change in the properties of [3H]NMS binding sites throughout the brain. In contrast, 2 h immobilization caused a significant increase in receptor affinity (Kd) without modification in the maximal number of receptors (Bmax) in several brain areas such as the caudate-putamen, cortical layers and CA1 field of the hippocampus, among others. These results, found even in animals killed immediately after the end of the immobilization sessions, suggest that immobilization stress induces supersensitivity of muscarinic receptors in certain cholinergic pathways in rat brain.     

Indorenate modifies a1-adrenergic and benzodiazepine receptor binding in the rat brain: an autoradiography study

Indorenate (5-methoxytryptamine-beta-methylcarboxylate) is a 5-HT1A receptor agonist that produces antihypertensive, anxiolytic, antidepressant and anticonvulsant effects. However, there is evidence suggesting that these effects could involve the activation of benzodiazepine (BZD) receptors but not the activation of a1-adrenergic receptors. The goal of this study was to analyse the effect of indorenate on a1-adrenergic and BZD receptor binding in specific rat brain areas by using in-vitro autoradiography. Coronal brain sections from male Wistar rats were used for labelling 5-HT1A (3H-8-OH-DPAT, 2 nM), a1-adrenergic (3H-prazosin, 2 nM) and BZD (3H-flunitrazepam, 2 nM) receptor binding in the presence or absence of indorenate (1 microM). Indorenate totally displaced 3H-8-OH-DPAT binding in all the brain areas evaluated. It decreased 3H-prazosin binding just in the frontal (30%) and sensorimotor (32%) cortices and in the thalamus (21%). Additionally, indorenate diminished 3H-flunitrazepam binding only in the cingulate (16%) and piriform (18%) cortices as well as in the dorsal raphe nucleus (18%). These results confirm that indorenate is a 5-HT1A ligand and suggest the possible participation of a1-adrenergic and BZD receptors in its pharmacological properties.     

Effects of acetylsalicylic acid on serotonin brain receptor subtypes

• 1.1. The lysine salt of acetyl salicylic acid (ASA) at a dose equivalent to 400 mg/kg of acetyl salicylic acid (ASA) was intraperitoneally administered in rats.
• 2.2. After 30 and 120min ASA did not modify the number of receptors nor the affinity of [³H]8-OH-DPAT binding sites in pons and cerebral cortex. On the other hand, the receptor number in the cortex membranes decreased significantly using [³H]ketanserin as ligand, while the receptor number in the pontine membranes did not change.
• 3.3. These data support the involvement of central 5-HT receptors in the mode of action of ASA.

     

Effects of selective 5-HT1A receptor antagonists on regional serotonin synthesis in the rat brain: an autoradiographic study with alpha-[14C]methyl-L-tryptophan.

The effects of acute and chronic administration of WAY100635 and WAY100135, serotonin (5-HT)1A antagonists, on 5-HT synthesis rates, calculated from the trapping of alpha-[14C]methyl-L-tryptophan (alpha-MTrp), were evaluated in the rat brain using autoradiography. In the acute treatment studies, WAY100635 (1 mg/kg) induced a significant increase in 5-HT synthesis in the median raphe nucleus and some nerve terminal structures (range between 18 and 53%), while WAY100135 (10 mg/kg) produced a significant decrease of synthesis, in the range between 16 and 33%, in the raphe magnus nucleus and several projection areas. The action of WAY100635 given acutely was likely a result of antagonist actions at the 5-HT1A somato-dendritic autoreceptors. WAY100135 probably acted acutely as a partial agonist. In the chronic treatment studies, WAY100635 (1 mg/kg/day) and WAY100135 (10 mg/kg/day) were administered for 7 days as s.c. injections once a day. Chronic treatment with both compounds significantly reduced the rate of 5-HT synthesis in the nerve terminal structures and produced a significant increase in the raphe nuclei. These treatments did not have any effect on the plasma free or total tryptophan.     

The effects of acute and chronic ethanol administration and its withdrawal on gamma-aminobutyric acid receptor binding in rat brain.

1 The effects of acute and chronic ethanol administration, and withdrawal on the binding of the inhibitory neurotransmitter, gamma-aminobutyric acid (GABA), was investigated in rat brain. 2 Acute ethanol (2 to 4 g/kg i.p. 30 min before removal of brain) produced an increase in the binding capacity of the low affinity GABA receptor binding site. 3 Following chronic ethanol administration (1 to 21 days), the GABA receptor binding characteristics were not altered. These results suggest a possible adaptation of GABA receptors to the continuous presence of ethanol at the GABA synapse. 4 During ethanol withdrawal, the affinity of the low affinity GABA receptor binding site was significantly lower than pair-fed controls at 8 and 16 h withdrawal. 5 These results suggest that GABA receptor sensitivity may play a role in some of the neuropharmacological effects of ethanol and in its withdrawal symptoms.     

Effect of flibanserin (BIMT 17), fluoxetine, 8-OH-DPAT and buspirone on serotonin synthesis in rat brain.

In male rats, the effects of the administration of the novel serotonergic agent flibanserin on the synthesis of 5-HT were evaluated in the frontal cortex (FC), hippocampus (Hip) and brainstem (Br). The selective serotonergic uptake blocker, fluoxetine, and two serotonin1A (5-HT1A) agonists, 8-hydroxy-2-(di-n-propylamino)tetraline (8-OH-DPAT) and buspirone, were used as reference compounds. The synthesis of 5-HT was assessed by measuring the accumulation of 5-hydroxytryptophan (5-HTP) after blockade of aromatic amino acid decarboxylase induced by m-hydroxybenzylhydrazine (NSD-1015), at 100 mg/kg i.p., 30 min before sacrifice. Flibanserin, 8-OH-DPAT and buspirone were given 15 min before NSD-1015, while fluoxetine 120 min before NSD-1015. In our experimental conditions, a different efficacy, expressed as percentage of maximal inhibition (Max) of 5-HTP accumulation, and a different potency, expressed in terms of minimal effective dose (MED), were observed in different brain areas with tested compounds. Flibanserin (1-32 mg/kg) decreased 5-HT synthesis with preferential activity in the FC, compared to the Hip and Br, both in terms of potency (MED=2 mg/kg in FC, 16 mg/kg in Hip and Br) and efficacy (Max=65% in FC, 44% in Hip and 29% in Br). Fluoxetine (1-30 mg/kg) decreased 5-HT synthesis with preferential activity in FC than in Hip and Br, only in terms of potency (MED=3 mg/kg in FC, 10 mg/kg in Hip and Br), this result being similar to that observed for flibanserin. In contrast, it showed greater efficacy both in FC and Hip (Max about 60%), than in Br (Max=49%). On the contrary, 8-OH-DPAT (0.3-3 mg/kg) decreased 5-HT synthesis with the same potency in all brain regions (MED=3 mg/kg) and showed the greatest efficacy in FC than in Hip and Br (Max=56% in FC, 49% in Hip and 40% in Br). Furthermore, buspirone (3-30 mg/kg), while inhibiting 5-HTP accumulation in all areas with the same efficacy (Max about 30%), seemed to have higher potency in Br than in FC and Hip (MED=3 mg/kg in Br, 10 mg/kg in FC and Hip). The results in terms of regional differences are discussed.     

Reduction in serotonin synthesis following acute and chronic treatments with paroxetine, a selective serotonin reuptake inhibitor, in rat brain: an autoradiographic study with alpha-[14C]methyl-L-tryptophan(2).

Serotonin (5-HT) synthesis rates were calculated on the basis of the assumption that trapping of alpha-[14C]methyl-L-tryptophan (alpha-[14C]MTrp) is directly related to brain 5-HT synthesis. In the first series of experiments, an acute intraperitoneal injection of paroxetine (10 mg/kg) produced a significant reduction in 5-HT synthesis in brain structures containing serotonergic cell bodies (the dorsal, median, and pallidum raphe nuclei), as well as in most projection areas: the ventral tegmental area, median forebrain bundle, hippocampus CA3 region, and nigrostriatal structures (substantia nigra, lateral and medial caudate nuclei). The reductions in the projection areas were greater (between 25 and 53%) than in those areas containing serotonergic cell bodies (between 18 and 23%). In the cerebral cortex, 5-HT synthesis rates were not modified by acute paroxetine treatment. In a second series of experiments, rats were treated with paroxetine (10 mg/kg/day, s.c., delivered by osmotic minipumps) for 14 days. There was a marked decrease (39-69%) in 5-HT synthesis in every structure examined. In conclusion, the present data suggest that the effects of paroxetine on 5-HT synthesis in the cerebral cortex are different from its effects in the cell body area of the brainstem.