Louise Grondin

[3H]paroxetine binding and serotonin content of rat cortical areas, hippocampus, neostriatum, ventral mesencephalic tegmentum, and midbrain raphe nuclei region following p-chlorophenylalanine and p-chloroamphetamine treatment.

Abstract: The agents p‐chlorophenylalanine (PCPA) and p‐chloroamphetamine (PCA) deplete brain serotonin (5‐HT) levels by two different mechanisms; PCPA inhibits the enzyme tryptophan hydroxylase, whereas PCA has a neurotoxic action on certain 5‐HT neurons. The parameters of [3H]paroxetine binding to homogenates prepared from the cerebral cortex of rats treated with PCPA, PCA, or saline vehicle were investigated. The tissue concentrations of 5‐HT and 5‐hydroxyindole‐3‐acetic acid (5‐HIAA) were also determined by HPLC in the same brain samples. After PCPA treatment, neither the maximum binding capacity (Bmax) nor the dissociation constant (KD) of [3H]paroxetine for the 5‐HT uptake recognition site differed from controls despite a substantial reduction in the concentration of 5‐HT and 5‐HIAA. In contrast, significant changes in both the Bmax and KD values were observed in the cerebral cortex of rats treated with PCA. Furthermore, [3H]paroxetine binding and tissue concentrations of 5‐HT and 5‐HIAA were measured in the following different regions of the rat brain: cingulate, parietal, and visual cortical areas; dorsal and ventral hippocampus; rostral and caudal halves of neostriatum; ventral mesencephalic tegmentum; and midbrain raphe nuclei region after administration of PCPA, PCA, or saline vehicle. There was an excellent correlation between regional 5‐HT levels and specific [3H]paroxetine binding in control and PCA‐treated rats although this correlation was lost after PCPA treatment. Under these conditions, the 5‐HT innervation remains unchanged whereas the concentration of 5‐HT and 5‐HIAA is greatly reduced. Thus, [3H]paroxetine binding appears to provide a reliable marker of 5‐HT innervation density within the mammalian CNS.

Effect of Ammonia on Brain Serotonin Metabolism in Relation to Function in the Portacaval Shunted Rat

Abstract: Four weeks following portacaval anastomosis (PCA) in the rat, severe liver atrophy, sustained hyperammonemia, and increased plasma and brain tryptophan are observed. Administration of ammonium acetate (NH4Ac) to rats with PCA precipitates severe signs of hepatic encephalopathy (HE) (loss of righting reflex progressing to loss of consciousness and ultimately deep coma). To evaluate the relationship between the deterioration of neurological status in HE and serotonin (5‐HT) metabolism, the levels of 5‐HT, its precursor 5‐hydroxytryptophan, and its major metabolite 5‐hydroxyindole‐3‐acetic acid (5‐HIAA) were measured by HPLC with ion‐pairing and electrochemical detection in three well‐defined areas of the cerebral cortex: anterior cingulate, piriform and entorhinal, and frontoparietal; as well as in the caudateputamen, the raphe nuclei, and the locus ceruleus in rats with PCA at different stages of HE, before and after injection of NH4Ac, as well as in sham‐operated controls. The results demonstrate increased 5‐HIAA/5‐HT ratios after PCA and NH4Ac loading, suggesting increased 5‐HT turnover in the brains of these animals. However, these changes do not appear to be related to the precipitation of coma as no significant difference in 5‐HT turnover was observed between precoma and coma stages of HE. Increased 5‐HT turnover in brain of shunted rats may be related to early symptoms of HE such as altered sleep patterns and disorders of motor coordination.

Heterogeneity of Cortical and Hippocampal 5‐HT1A Receptors: A Reappraisal of Homogenate Binding with 8‐[3H]Hydroxydipropylaminotetralin

Abstract: The selective serotonin (5‐HT) agonist 8‐hydroxydipropylaminotetralin (8‐OH‐DPAT) has been extensively used to characterize the physiological, biochemical, and behavioral features of the 5‐HT1A receptor. A further characterization of this receptor subtype was conducted with membrane preparations from rat cerebral cortex and hippocampus. The saturation binding isotherms of [3H]8‐ OH‐DPAT (free ligand from 200 pM to 160 nM) revealed high‐affinity 5‐HT1A receptors (KH= 0.7–0.8 nM) and lowaffinity (KL= 22–36 nM) binding sites. The kinetics of [3H]8‐OH‐DPAT binding were examined at two ligand concentrations, i.e., 1 and 10 nM, and in each case revealed two dissociation rate constants supporting the existence of high‐ and low‐affinity binding sites. When the high‐affinity sites were labeled with a 1 nM concentration of [3H]8‐ OH‐DPAT, the competition curves of agonist and antagonist drugs were best fit to a two‐site model, indicating the presence of two different 5‐HT1A binding sites or, alternatively, two affinity states, tentatively designated as 5‐HT1AHIGH and 5‐HT1ALOW. However, the low correlation between the affinities of various drugs for these sites indicates the existence of different and independent binding sites. To determine whether 5‐HT1A sites are modulated by 5′‐guanylylimidodiphosphate, inhibition experiments with 5‐HT were performed in the presence or in the absence of 100 μM 5′‐guanylylimidodiphosphate. The binding of 1 nM [3H]8‐OH‐DPAT to the 5‐HT1AHIGH site was dramatically (80%) reduced by 5′‐guanylylimidodiphosphate; in contrast, the low‐affinity site, or 5‐HT1ALOW, was seemingly insensitive to the guanine nucleotide. The findings suggest that the high‐affinity 5‐HT1AHIGH site corresponds to the classic 5‐HT1A receptor, whereas the novel 5‐HT1ALOW binding site, labeled by 1 nM [3H]8‐OH‐DPAT and having a micromolar affinity for 5‐HT, may not belong to the G protein family of receptors. To further investigate the relationship of 5‐HT1A sites and the 5‐HT innervation, rats were treated with p‐chlorophenylalanine or with the neurotoxin p‐chloroamphetamine. The inhibition of 5‐HT synthesis by p‐chlorophenylalanine did not alter either of the two 5‐HT1A sites, but deafferentation by p‐chloroamphetamine caused a loss of the low‐affinity [3H]8‐OH‐ DPAT binding sites, indicating‐that these novel binding sites may be located presynaptically on 5‐HT fibers and/or nerve terminals.

Distribution of catecholamines, serotonin, and their major metabolites in the rat cingulate, piriform-entorhinal, somatosensory, and visual cortex: a biochemical survey using high-performance liquid chromatography

The catecholamines noradrenline (NA), dopamine (DA), adrenaline (AD), the indoleamine 5-hydroxytryptamine (5-HT; serotonin), as well as some of their major metabolites were assayed by high-performance liquid chromatography (HPLC) with electrochemical detection, in four well-defined areas of the rat cerebral cortex: anterior cingulate (CIN;Cg1 and Cg3), piriform and entorhinal (PiEn), hind-limb primary somatosensory (SSC;HL) and primary visual (VIS; Oc1M and Oc1B). The concentrations of NA and that of its main metabolite 3-methoxy-4-hydroxyphenylglycol were highest in PiEn, had intermediate values in CIN and were lowest for SSC and VIS cortices. The DA levels were also highest in PiEn, intermediate in CIN, while the lowest values were in SSC and VIS cortices. The different DA/NA ratios support the hypothesis that they are indeed independent neurotransmitters. In addition, the levels of 3,4-dihydroxyphenylacetic acid, homovanillic acid and 3-methoxytyramine paralleled the distribution of DA, thus confirming the presence of release sites, even in regions in which the low levels of this catecholamine could be interpreted simply as the precursor of NA. Traces of AD were detected in all the regions examined. The 5-HT contents, as well as that of its precursor 5-hydroxy-I-tryptophan and that of its metabolite 5-hydroxyindole-3-acetic acid were also found to be non-homogenous, with the highest levels measured in the PiEn and CIN regions.

Specific [3H]SCH23390 Binding to Dopamine D1 Receptors in Cerebral Cortex and Neostriatum: Evidence for Heterogeneities in Affinity States and Cortical Distribution

The tritiated antagonist SCH23390 was used to identify dopamine D1 receptors in the cerebral cortex and neostriatum. The kinetic properties of binding were investigated in parallel experiments with membrane preparations from both tissues. The densities of receptors (Bmax) and the dissociation constants (KD) were determined from saturation curves, and the specificity of binding verified in competition experiments using agonists and antagonists. The cortical D1 receptor displays the same pharmacological selectivity (including stereospecificity) and kinetic properties as the neostriatal D1 receptor. From both the dissociation kinetics by dilution and the competition curves, it could be established that there is an heterogeneity of binding, probably due to high‐ and low‐affinity states. Endogenous dopamine, 4‐hydroxy‐3‐methoxyphenylacetic acid, 3,4‐dihydroxyphenylacetic acid, and 3‐methoxytyramine contents, as well as D1 receptor distribution, were measured for the neostriatum and four localized cortical areas: anterior cingulate. primary somatosensory, primary visual, and piriform‐entorhinal. For the regions examined, the distribution of D1 receptors is heterogeneous, but correlates very well (r > 0.98) with the endogenous levels of dopamine and its major metabolites.

Effects ofp-chlorophenylalanine on cortical monoamines and on the activity of noradrenergic neurons

The catecholamines noradrenaline, dopamine, adrenaline, the indoleamine 5-hydroxy-tryptamine (5-HT; serotonin), and some of their major metabolites were assayed, using high performance liquid chromatography (HPLC), in the neocortex of normal rats as well as in animals in which 5-HT synthesis had been inhibited withp-chlorophenylalanine. Besides important depletions in serotonin and in 5-hydroxyindole-3-acetic acid, noradrenaline levels were significantly reduced, but the content in 3-methoxy-4-hydroxyphenylglycol was increased, indicating an augmented utilization of this amine. The levels of dopamine and 3-methoxytyramine were also reduced, although homovanillic acid and 3,4-dihydroxyphenylacetic acid levels remained constant. The spontaneous unitary activity of identified noradrenergic neurons in the Locus coeruleus was increased, indicating an hyperactivity of this system. These results can be interpreted in relation to functional interactions between the catecholamines and serotonin; i.e.: a decrease in endogenous serotonin results in the loss of a negative feedback control of noradrenaline release.

Adrenergic receptor and catecholamine distribution in rat cerebral cortex: binding studies with [3H]prazosin, [3H]idazoxan and [3H]dihydroalprenolol☆

The tritiated adrenergic antagonists [3H]dihydroalprenolol ([3H]DHA; beta-receptors), [3H]prazosin ([3H]PRZ; alpha 1-receptors), and [3H]idazoxan ([3H]IDA; alpha 2-receptors) were used to determine the distribution of these sites in 5 defined areas of the adult rat cerebral cortex. The highest density of [3H]PRZ binding was found in the prefrontal cortex, with a lower and homogeneous distribution for the frontal, parietal, occipital and temporal areas. The [3H]IDA binding sites were fairly uniform for all areas, except for the temporal cortex where it was very dense. In contrast, beta-adrenoceptors labelled by [3H]DHA were very homogeneous for all the regions examined. The functional significance of the distribution of alpha 1, alpha 2 and beta-adrenoceptors is discussed in relation to the catecholamine innervation and monoamine contents measured by high performance liquid chromatography.

[3H]Paroxetine binding and serotonin content of rat brain : absence of changes following antidepressant treatments

The high affinity binding of [3H]paroxetine was measured in rat cerebral cortex following chronic treatment (21 days) with imipramine (5 mg/kg), trimipramine (5 mg/kg) and fluoxetine (2 mg/kg), in adult (3-4 months) or neonatal (7 days of age) rats. Tissue concentrations of serotonin and of its metabolite 5-hydroxyindole-3-acetic acid were also determined by high-performance liquid chromatography in cingulate cerebral cortex, rostral neostriatum, hippocampus and midbrain raphe nucleus region. No differences were found in any of the parameters of [3H]paroxetine binding after antidepressant administration, in either adult or neonatal animals. In addition, endogenous serotonin and 5-hydroxyindole-3-acetic acid levels were not different from control values in any of the regions examined. The present study shows that the serotonin uptake recognition site is resilient to changes after chronic treatment with therapeutic doses of antidepressants, and emphasizes the potential usefulness of uptake site ligands as markers to quantify innervation densities within the brain.

Distribution of monoamines and metabolites in rabbit neostriatum, hippocampus and cortex.

The monoamines noradrenaline (NA), dopamine (DA), adrenaline (AD) and 5-hydroxytryptamine (5-HT) were assayed in the putamen (PUT), the lateral (lCAU) and medial (mCAU) portions of the caudate, the dorsal (dHIP) and ventral (vHIP) hippocampus, as well as in four cortical areas, i.e., anterior cingulate (CIN), entorhinal-piriform (EnPi), sensorimotor (SSC; somatosensory) and primary visual (VIS). The use of an HPLC procedure enabled us to perform these measurements in microdissected samples and to assay as well monoamine metabolites. The DA levels were highest in the neostriatum, moderate in the EnPi and CIN and very low in the SSC, VIS and hippocampus. The distribution of NA was more uniform, although higher concentrations were measured in the neostriatum, hippocampus and EnPi. The largest amounts of 5-HT were in the EnPi, while moderate concentrations were found in the other regions. The ratios between the neurotransmitters and their metabolites were used as an index of turnover and indicate that the terminal fields of the monoamine systems are heterogenous within the neostriatal, hippocampal and cortical subdivisions.

Alpha-1 and alpha-2 adrenoceptor binding in cerebral cortex: Role of disulfide and sulfhydryl groups

The triated adrenergic antagonists Prazosin ([3H]PRZ) and Idazoxan ([3H]IDA, or RX-781094) bind specifically and with high affinity to α1 and α2-adrenoceptors respectively, in membrane preparations from cerebral cortex. Saturation experiments performed to determine the density of receptors and the dissociation constant (Kd) were analyzed by the methods of Eadie Hofstee, iterative modelling, and the procedure of Hill, while the specificity of the labelling was verified by displacement experiments. Since receptors are proteins, we examined the role of disulfide (−SS−) bridges and sulfhydryl (−SH) groups in the specific combination of [3H]PRZ and [3H]IDA to the α1 and α2-adrenoceptors. Pretreatment of the membranes with the −SS− reactive DL-dithiothreitol (DTT) or the alkylating agent N-ethylmaleimide (NEM), alone or in combination, decreased specific binding of both ligands, with only minor changes in the non-specific counts. The [3H]IDA binding (α2-sites) was more sensitive to both DTT and NEM than the [3H]PRZ sites (α2-adrenoceptors), and the initial changes induced by alkylation of the α2-site were due to an important decrease in the affinity for [3H]IDA, as judged by the increase in theKd. This modulation in the affinity caused by alkylation of a thiol group could explain the higher potency of the blocking agent tetramine disulfide benextramine at the α2-site. The results provide evidence for the participation of −SS− and −SH groups in the binding site of α1 and α2-adrenoceptors in the cerebral cortex.