Karen M. Dewar

Serotonin 5-HT1 and 5-HT2 receptors in adult rat brain after neonatal destruction of nigrostriatal dopamine neurons: a quantitative autoradiographic study

Neonatal destruction of nigrostriatal dopamine neurons by cerebroventricular injection of 6-hydroxydopamine (6-OHDA) results in a serotonin (5-HT) hyperinnervation of the rostral neostriatum in adult rat. Quantitative ligand-binding autoradiography was used to compare the density of various 5-HT receptor subtypes in the adult brain of control and neonatally 6-OHDA-lesioned rats. 5-HT1A, 5-HT1B, 5HT1nonAB and 5-HT2 sites were labeled with [3H]8-OH-DPAT, [125I]cyanopindolol, [3H]5-HT and [125I]DOI, respectively, and measured in the rostral and caudal halves of neostriatum and selected forebrain or midbrain regions. 5-HT1A binding, measured after 6 months, was unchanged in all regions examined including the dorsal raphe nucleus. Three months after the lesion, 5-HT1B binding was increased throughout the neostriatum (30%), but also in the substantia nigra (50%) and globus pallidus (30%), suggesting an up-regulation and an increased axonal transport of these receptors in neostriatal projection neurons. 5-HT1nonAB binding was also increased throughout the neostriatum (40%) and in the substantia nigra (50%), but unchanged in the globus pallidus, as if this up-regulation preferentially involved striatonigral as opposed to striatopallidal neurons. 5-HT2 binding showed an even greater increase (60%), which was restricted to the rostral half of neostriatum and also seemed imputable to an up-regulation as heteroreceptors. Even though the exact cause(s) of these receptor increases could not be determined, their anatomical distribution suggested that they were somehow related to the initial dopamine denervation in the case of the 5-HT1B and 5-HT1nonAB receptors, and more tightly linked to the 5-HT hyperinnervation in the case of the 5-HT2 receptors. Such receptor changes could participate in adaptive mechanisms implicating other transmitters and behavioral disturbances observed in this particular experimental model. Interestingly, they could also account for an enhancement of neostriatal 5-HT function even in a condition where extracellular levels of 5-HT apparently remain normal because of increased uptake.

ELEVATION OF DOPAMINE D2 BUT NOT D1 RECEPTORS IN ADULT RAT NEOSTRIATUM AFTER NEONATAL 6-HYDROXYDOPAMINE DENERVATION

Monoamine levels and the binding properties of [3H]SCH23390, a D1-specific ligand, and [3H]raclopride, a D2-specific ligand, were measured in the rostal and caudal neostriatum to investigate the fate of dopamine receptors following bilateral cerebroventricular injection of 6-hydroxydopamine in 3-day-old rats. After survival times of 15, 30 or 90 days, measurement of monoamine levels and of [3H]SCH23390 binding were also obtained from the cerebral cortex. At all three survival times, dopamine content was reduced by more than 90% of control values in both the rostral and caudal neostriatum; in cerebral cortex, the dopamine depletion was less profound (80%) and noticeable only after 1 and 3 months. In the rostral but not the caudal neostriatum, serotonin and 5-hydroxyindoleacetic acid concentrations were markedly increased at 1 and 3 months; cortical serotonin also was augmented at 3 months. There were no changes in neostriatal [3H]SCH23390 binding at any of the survival times, but a transient elevation occurred in the cortex at 1 month. In the rostral but not the caudal neostriatum, [3H]raclopride binding showed a slight elevation at 1 month and a further, highly significant increase at 3 months. As measured in individual rats, this increase in [3H]raclopride binding was linearly correlated with the increase in serotonin turnover (ratio of 5-hydroxyindoleacetic acid/serotonin). Such an up-regulation of D2 receptors, restricted to the rostral neostriatum which was also the site of a serotonin hyperinnervation, was probably indicative of a serotonin control on the expression of D2 receptors after dopamine denervation.

Review article Effects of denervation and hyperinnervation on dopamine and serotonin systems in the rat neostriatum: implications for human Parkinsons disease

Abstract The research on central synaptic neurotransmission has greatly benefited from the use of the neurotoxin 2,4,5-trihydroxyphenylethylamine, or 6-hydroxydopamine (6-OHDA), that destroys catecholamine-containing neuronal cell bodies and nerve terminals. Refinements in the use of this neurotoxin led to the use of dopamine-denervated animals as models of human Parkinsons disease, in which the loss of dopaminergic neurons is a prominent feature. Here we review structural, pharmacological, and biochemical studies carried out in the adult and neonatal 6-OHDA lesioned animals. These models have become useful and interesting paradigms to examine alterations in the expression of receptors and in their sensitivity to agonist drugs; some of these modifications may underlie the altered responsiveness of the dopamine-lesioned animals to dopamine, but also to other compounds, including serotoninergic drugs. We have also reviewed studies of amino acids as well as of monoamine metabolism and of uptake mechanisms that may underlie some of the behavioural alterations in these models that have become relevant for our understanding of the sprouting and plastic properties of spared neurons, and of the alternate neuronal projections that replace lesioned terminals, enabling compensatory adaptations. Although 6-OHDA-lesioned animals, that display some biochemical characteristics of Parkinsons disease in humans, do not express all of the neurological features exhibited by patients, the increasing knowledge that can be obtained from studies in simplified experimental models will undoubtedly lead to the development of innovative drugs and other replacement therapies for degenerative brain diseases.

Effects of chronic lithium treatments on central dopaminergic receptor systems: G proteins as possible targets

Numerous biochemical and electrophysiological studies have proposed a role for dopamine (DA) in the therapeutic efficacy of lithium (Li+) salts. The effects of ex vivo chronic Li+ treatments on neostriatal DA receptors, as well as on the G protein adenylyl cyclase complex and on tissue cAMP levels were investigated in adult rats. The animals were administered LiCl in their drinking water (1 g/l) for varying periods of time, i.e. 1, 15 and 28 days. After sacrifice by decapitation, their brains were removed and the neostriatum dissected out to assay DA receptors and adenylyl cyclase activity. The antagonists [3H]SCH23390 and [3H]raclopride were employed to label D1 and D2 receptors, respectively. Chronic Li+ treatments did not modify the saturation binding of either ligand. However, competition studies of the same antagonists by DA revealed biphasic curves, and the inhibition constant of the high-affinity site was significatively increased after chronic Li+. The data suggest an alteration in the coupling efficacy between G proteins and DA receptors. Moreover, chronic (28 day) Li+ treatment, but not a 1 day Li+ administration, lead to a reduction of the GTP-induced and DA-sensitive adenylyl cyclase activity, without changes in the basal activity or in forskolin-induced cAMP production. The results demonstrate that chronic Li+ treatments diminish neostriatal dopaminergic activity, probably through a direct action on the G protein itself. The underlying mechanisms do not appear to involve modifications in either the D1 or the D2 receptor primary ligand recognition sites, but may represent alterations in both the coupling process and the capacity of the G proteins, once activated, to stimulate adenylyl cyclase.

[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.

Amino acid levels and γ-aminobutyric acidA receptors in rat neostriatum, cortex, and thalamus after neonatal 6-hydroxydopamine lesion

Abstract: The amino acid γ‐aminobutyric acid (GABA) is the main inhibitory neurotransmitter in brain, and GABAergic neurons have been proposed to play a major role in basal ganglia physiology. In the neostriatum (caudate putamen), medium‐sized aspiny interneurons, as well as neostriatal output neurons that project to several brain regions, use GABA as their neurotransmitter. Dopamine fibers arising from the substantia nigra represent a major input to the neostriatum where, besides their classic neurotransmitter role, they are seemingly involved in the regulation of amino acid neurotransmitter release. To further characterize the nature of some of the amino acid/dopamine interactions, selective dopaminergic deafferentations were produced in neonatal rats (3 days postnatal) by intraventricular administration of the neurotoxin 6‐hydroxydopamine (6‐OHDA); the noradrenergic neurons were protected by prior administration of desmethylimipramine. After a 3‐month survival, levels of catecholamines, indoleamines, and amino acids were determined in cingulate cortex, thalamus, and neostriatum. In addition, GABAA receptors were measured in membrane preparations from these three regions, using the specific agonist [3H]muscimol. In the 6‐hydroxydopamine‐lesioned rats, levels of dopamine and its metabolites homovanillic acid, 3,4‐dihydroxyphenylacetic acid, and 3‐methoxytyramine were decreased, as expected, in cortex and neostriatum, but remained unmodified in thalamus. In all three regions, serotonin content was increased; its metabolite, 5‐hydroxyindole‐3‐acetic acid, was also elevated, but only in cortex and neostriatum. The levels of GABA were increased in neostriatum and thalamus, but remained unmodified in cortex. Glycine was increased in all three regions examined. There were also increases of phosphatylethanolamine and serine in thalamus, and of aspartic acid and alanine in neostriatum. The density of GABAA binding sites was increased in neostriatum, but remained unchanged in cortex and thalamus. The changes in amino acid levels and [3H]muscimol binding sites induced by a neonatal 6‐hydroxydopamine treatment differ from those found after similar lesions in adult animals, possibly because of the plastic and synaptic rearrangements that can still occur during early postnatal development. The present results also demonstrate that adaptations occur in response to a dopaminergic deafferentation at an early age and that these exhibit a regional specificity.

Specific [3H]SCH23390 Binding to Dopamine D1 Receptors in Cerebral Cortex and Neostriatum: Role of Disulfide and Sulfhydryl Groups

Abstract: Receptor binding studies were performed in cerebral cortex (CTX) and neostriatum (CPU; caudate–putamen) using the dopamine D1 antagonist [3H]SCH23390. Because receptors are of protein nature, we examined the role of disulfide bonds (–SS–) and sulfhydryl groups (–SH) in the specific binding of [3H]SCH23390. Furthermore, membrane preparations contain a certain amount of lipid, so that treatments with –SH and –SS– reagents could determine whether the fixation of the radioligand was to protein or to the lipid moiety. Pretreatment of CTX and CPU membranes with dithioerythritol, l‐dithiothreitol, or 5,5′‐dithiobis(2‐nitro‐benzoic acid), as well as with the alkylating agent N‐ethyl‐maleimide, produced dose‐dependent decreases of specific [3H]SCH23390 binding in membrane preparations from both tissues. These changes were not reversible after up to two washes, but could be prevented in part if the treatments were performed in the presence of dopamine. Additional protection experiments were conducted with (+)‐ and (−)‐butaclamol, as well as with (+)‐ and (−)‐SKF38393. A series of saturation experiments (with pretreated membranes in the absence of reactives) demonstrated that the alkylation of –SS– groups reduced specific [3H]SCH23390 binding mainly through an affinity change, but l‐dithiothreitol and 5,5‐dithiobis(2‐nitrobenzoic acid) decreased the number of binding sites. The affinity of the receptor to agonists was examined with the two enantiomers of SKF38393; the inhibition curves showed that residual binding was not affected and stereospecificity was conserved. The present results provide evidence for the participation of both –SS– and –SH groups in the recognition site of the dopamine D1 receptor in both the CTX and the CPU.

Comparative biochemical pharmacology of central nervous system dopamine D1 and D2 receptors

The biochemical properties of central nervous system (CNS) dopamine (DA) D1 and D2 receptors were examined using the specific antagonists [3H]SCH23390 and [3H]raclopride, respectively. There is a different participation of sulfhydryl (-SH) and disulfide (-SS-) groups in the binding site and/or coupling to second messenger systems of D1 and D2 receptors. The ionioc studies with [3H]SCH23390 showed slight agonist and antagonist affinity shifts for the D1 receptor. On the other hand, the D2 receptor is very sensitive to cations; even if lithium and sodium influence specific [3H]raclopride binding in a similar manner, there appear to be quantitative differences between these two ions that cannot be explained by surface charge mechanisms. The distribution of D1 and D2 receptors was heterogenous in both species, with the greatest densities in the neostriatum, where the highest concentrations of DA and metabolites were measured. Regions with low endogenous DA content (cerebral cortex and hippocampus) had lower densities of DA receptors. Furthermore, these binding sites were differentially localized within the various regions, and there were substantially more D1 than D2 receptors. The functional significance and heterogeneities in the distribution of D1 and D2 receptors can be related to dopaminergic innervation and turnover. *** DIRECT SUPPORT *** A07DG003 00017

Endogenous homovanillic acid levels differ between rat and rabbit caudate, hippocampus, and cortical regions.

Endogenous dopamine (DA) levels and its metabolites 3,4-dihydroxyphenylacetic acid (DOPAC), 3-methoxytyramine (3MT) and homovanillic acid (HVA) were measured by high-performance liquid chromatography in the entorhinal-piriform (EnPi), cingulate (CIN), sensorimotor (SSM) and visual (VIS) cortices as well as is the caudate (CAU) and hippocampus (HIP) of Sprague-Dawley (SD) rats and New Zealand (NZ) rabbits. The DA, DOPAC and 3MT contents were similar in both species. The HVA levels however, although they followed DA distribution, were several-fold higher in NZ rabbits than in SD rats for all cortices, HIP and CAU. In addition, total metabolite contents and DA turnove (estimated from ‘DA metabolite/DA’ ratios) were significantly higher in NZ rabbits than in SD rats, suggesting an increased release and/or metabolism in the former species. The HVA/DA ratios were much higher for NZ rabbit regions than for SD rats, indicating an increased DA release in the former species since the DOPAC/DA ratios (index of intraneuronal degradation) were similar.