The quest for biological correlates of social phobia: an interim assessment.

OBJECTIVE: The primary goal of this review was to assess critically the literature concerning the ongoing search for possible biological correlates of social phobia. METHODS: In addition to manual searches, Medline, Current Contents and Psych Info databases were searched for relevant publications. RESULTS: On the evidence of an extensive body of research, so far biological correlates of social phobia remain elusive. Furthermore, the majority of studies reveal by default that the neurobiological functioning of social phobics is very much like that of normal control subjects. CONCLUSION: The conceptual and methodological foundations underpinning the current research programme are discussed critically. Its main weaknesses were found to be: lack of theory to guide research and aid the interpretation of results, static comparisons between subject groups and analysis oblivious to great individual variations. Possibilities of alternative approaches to study the neurobiology of social phobia are raised. Among others, continuous and situation-specific measurement, subjects used as their own controls and neurobiological correlates of clinical improvement following psychotherapy are considered.

Alterations in serotonin receptors in the neostriatum of weaver mutant mice.

Mice that carry the autosomal recessive gene weaver show a distinctive loss of nigrostriatal dopamine innervation, with the greatest deficits in the dorsal caudate-putamen and almost complete sparing in the nucleus accumbens and ventral caudate. In addition to loss of dopamine in this model, it has recently been shown that markers of serotonin (5-hydroxytryptamine, 5-HT) innervation including 5-HT content, synaptosomal uptake of [3H]5-HT and [3H]citalopram binding were elevated in the dorsal neostriatum of the weaver mutant mouse. Using quantitative autoradiography of specific ligands for dopamine and 5-HT uptake sites as well as serotonin 5-HT1 and 5-HT2A receptors, we found an increased density of 5-HT uptake sites and 5-HT1 receptors restricted to the dorsal portion of the neostriatum of the weaver mouse. In contrast, 5-HT2A receptors were increased in both the dorsal and ventral portions of the rostral neostriatum as well as the nucleus accumbens. The behavioural and functional relevance of these receptor changes is unclear, although, adaptations in 5-HT may play a role in certain aspects of spontaneous behaviour in the weaver mutant mouse.

Apparent dopamine D1 and D2 receptors in the weaver mutant mouse: receptor binding and coupling to adenylyl cyclase.

Weaver mutant mice have a selective degeneration of the nigrostriatal dopamine pathway arising between 7-21 days after birth. The goal of this study was to investigate the effects of this mutation on different parameters of the nigrostriatal and mesolimbic dopamine system: apparent D1 and D2 receptor binding sites as well as their signal transduction pathway. Using quantitative autoradiography of ligands for dopamine D1, D2 receptors and the dopamine uptake site, we found a significant loss in apparent D1 receptor binding sites throughout the neostriatum, significant increase of apparent D2 receptor binding in the dorsal aspect of the neostriatum, and almost complete loss of DA uptake sites in these regions of the weaver mouse. In contrast to the neostriatum, the density of dopamine receptors and uptake sites in the nucleus accumbens of the weaver mouse did not differ from controls. Despite alterations in the binding of apparent D1 and D2 receptors, there was no significant difference in either basal, DA stimulated or GTPgammaS stimulated cAMP production. These findings suggest the down-regulation of apparent D1 receptor binding sites reported in this model, probably does not reflect an important physiological mechanism through which these animals compensate for loss of dopamine innervation.

Effects of denervation and hyperinnervation on dopamine and serotonin systems in the rat neostriatum: implications for human Parkinson's disease.

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 Parkinson's 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 Parkinson's 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.

Interaction between dopamine and glutamate receptors following treatment with NMDA receptor antagonists.

Interactions between dopamine and glutamate neurotransmission have been reported to play an important role in a number of different systems. We were interested in examining the effects of sub-chronic treatment with NMDA receptor antagonists (dizocilpine [MK-801], and 3-carboxy-piperazin-propyl phosphonic acid [CPP]) on dopamine D(1)-like, dopamine D(2)-like, as well as glutamate receptors of the NMDA and AMPA receptor subtypes in the neostriatum and substantia nigra of rats that had received a massive dopamine denervation at 3 days of age. Using quantitative ligand binding autoradiography, we demonstrated that the two NMDA receptor antagonists did not have different profiles of action. Furthermore, while we found a significant negative relationship between NMDA receptors and dopamine receptors (both dopamine D(1)-like and D(2)-like receptor subtypes) in the neostriatum, AMPA receptors were positively correlated with dopamine D(1)-like binding sites in all regions investigated. These findings suggest that the interrelationship between dopamine and glutamate receptors is highly controlled and that the nigrostriatal dopamine systems play an important role in this interaction.

Alterations in the turnover rate of dopamine D1 but not D2 receptors in the adult rat neostriatum after a neonatal dopamine denervation.

Adult rats that were treated with intracerebral ventricular injection of 6-hydroxydopamine (6-OHDA) as neonates exhibit a profound loss of nigrostriatal dopamine innervation in addition to a variety of other neurochemical and anatomical changes, including alterations in the number of neostriatal D1 and D2 receptor binding sites. In the present study, the turnover of neostriatal dopamine D1 and D2 receptors was measured in rats previously treated with 6-OHDA or ascorbic acid vehicle as neonates at various time intervals after peripheral N-ethoxycarbonyl-2-ethoxy-1, 2-dihydroquinoline (EEDQ, 10 mg/kg) administration. Dopamine receptors were labelled with [3H]SCH23390 (D1) and [3H]raclopride (D2), while the degree of dopamine denervation was assessed by the measurement of neostriatal dopamine, homovanillic acid and dihydroxyphenylacetic acid content. Two days after acute EEDQ treatment, the maximal binding capacity (Bmax) of [3H]SCH23390 and [3H]raclopride binding was significantly decreased to 58 and 32% of control values, respectively, without any significant alteration in their equilibrium dissociation constants (Kd). A time-dependent increase in the density of [3H]SCH23390 and [3H]raclopride binding was observed in both treatment groups following a single dose of EEDQ. The rate of recovery of D1 receptors was significantly slower in the 6-OHDA-lesioned animals as compared to controls with a half-life of 103 compared to 53 h, respectively. No differences were observed in the rate of recovery of D2 receptors in these two treatment groups. These data are consistent with the findings of decreased expression of D1 receptors in neonatal 6-OHDA-lesioned rats owing to decreased receptor synthesis, and further suggest that in this model the up-regulation of D2 receptors is a result of a post-transcriptional mechanism, such as an increased rate of post-synthetic maturation.

Excitotoxic lesions of the prefrontal cortex reduce dopamine D1-like receptors in the ventral tegmental area.

Ventral mesencephalic dopamine D1-like receptors were quantified in brains of male rats ten days after unilateral microinjections of ibotenic acid (2 or 10 microg/microl) or its vehicle into the medial prefrontal cortex. The density of dopamine D1-like receptors was reduced by more than 40% in the ipsilateral ventral tegmental area (both doses) and by 15% (low dose) and 44% (high dose) in the contralateral side; no significant reduction was observed in the substantia nigra. These results suggest that a significant number of ventral tegmental D1-like receptors are localized on afferent terminals from the medial prefrontal cortex.

Dopamine receptor alterations correlate with increased GABA levels in adult rat neostriatum: effects of a neonatal 6-hydroxydopamine denervation.

The effects of neonatal intracerebroventricular 6-hydroxydopamine (6-OHDA) injection on the densities of dopamine (DA) receptors and GABA levels were determined in the rostral neostriatum of adult rats. Measurement of GABA turnover indicated that increased tissue GABA in the DA-lesioned neostriatum is a consequence of higher GABA synthesis rate (205%). Binding experiments with [3H]SCH23390 (D1 receptors) and [3H]raclopride (D2 receptors) point to a correlation between tissue GABA content and altered DA receptors. Three months after the lesion there was a 27% decrease in D1 receptors and a 22% increase in D2 receptors. In control neostriatum, GABA levels were inversely related to D2 receptors and this relationship was reversed after 6-OHDA treatment. In contrast, the positive correlation between GABA and D1 receptors remained unchanged after the lesion. Irreversible blockade of DA receptors by N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) decreased both D1 and D2 sites (73-87%) in both control and lesioned neostriatum, but increased GABA levels by 25% only in animals which have received 6-OHDA just after birth. Following acute inhibition of DA synthesis or of DA catabolism, GABA levels remained unchanged. The present results indicated that DA depletion by itself is not the cause for the increase in GABA levels. The augmented GABAergic activity following neonatal 6-OHDA is seemingly influenced primarily by DA receptor status; presumably, changes in D2 receptor properties during maturation may be a principal cause for an increase in neostriatal GABA content.

Altered dopamine and serotonin metabolism in the dopamine-denervated and serotonin-hyperinnervated neostriatum of adult rat after neonatal 6-hydroxydopamine.

3,4-Dihydroxyphenylalanine (L-DOPA), 5-hydroxy-l-tryptophan (5-HTP), dopamine (DA), 5-hydroxytryptamine (5-HT) and their metabolites were measured in the rostral neostriatum of adult rats neonatally lesioned with 6-hydroxydopamine (6-OHDA) after treatment with NSD-1015 (aromatic L-amino acid decarboxylase inhibitor), pargyline (monoamine oxidase inhibitor) or probenecid (blocker of acidic metabolite efflux). Binding experiments with DA and 5-HT transporter ligands allowed us to relate observed changes to the number of DA and 5-HT terminals. One and 3 months after the lesions were made, DA, 3,4-dihydroxyphenylacetic acid (DOPAC)- and homovanillic acid (HVA)-content as well as [3H]N-[1-(2-benzo(b)-thiophenyl)cyclohexyl]piperidine ([3H]BTCP) binding were lowered to 1% to 2% of control values, reflecting a permanent DA denervation. After 1 month, 5-HT content was increased by 96% and 5-hydroxyindole-3-acetic acid (5-HIAA) was increased by 50% in the presence of control levels of [3H]citalopram binding, suggesting that there was an increased amount of 5-HT per neostriatal 5-HT terminal. At 3 months, 5-HT content had increased by 205%, 5-HIAA remained increased by 50% and [3H]citalopram binding had reached 170% of control values, indicating a persistent increase in 5-HT content within an excessive number of 5-HT terminals (hyperinnervation). At both survival times, neostriatal L-DOPA accumulation after NSD-1015 was low, but ratios of L-DOPA to DA and of L-DOPA to [3H]BTCP were greatly elevated. 5-HTP accumulation was increased, but ratios of 5-HTP to 5-HT were lower than control, despite unchanged ratios of 5-HTP to [3H]citalopram.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Changes of D1 and D2 receptors in adult rat neostriatum after neonatal dopamine denervation: quantitative data from ligand binding, in situ hybridization and iontophoresis.

The specific binding of [3H]SCH23390 to D1 and of [3H]raclopride to D2 dopamine receptors was measured by autoradiography in the rostral and caudal halves of neostriatum and in the substantia nigra of adult rats subjected to near total destruction of nigrostriatal dopamine neurons by intraventricular 6-hydroxydopamine soon after birth. Three months after this lesion, [3H]SCH23390 binding (D1 receptors) was slightly but significantly decreased in the rostral neostriatum (22%), but unchanged in its caudal half and in the substantia nigra. In contrast, [3H]raclopride binding (D2 receptors) was considerably increased throughout the neostriatum (10-40%), while markedly decreased in the substantia nigra (80%). In the rostral neostriatum, there were no parallel changes in D2 receptor messenger RNA levels, as measured by in situ hybridization on adjacent sections. Caudally, however, slight but significant increases in D2 messenger RNA could be observed (10-20%). As assessed by quantitative iontophoresis, there was a marked enhancement (63%) of the inhibitory responsiveness of spontaneously firing units in the rostral neostriatum to dopamine and the D1 agonist, SKF38393, in neonatally lesioned compared to control rats. On the other hand, responsiveness to PPHT, a potent D2 agonist, appeared to be unchanged. Such opposite changes in the number of D1 and D2 binding sites, dissociated from the expression of D2 receptor messenger RNA and from the sensitivity to dopamine and D1 and D2 agonists, suggested independent adaptations of these various parameters following the neonatal dopamine denervation of neostriatum. They also provided further evidence for mechanisms other than the dopamine innervation in the control of the expression of neostriatal D2 receptor messenger RNA during ontogenesis, and emphasized that the effects of dopamine and its D1 and D2 agonists in neostriatum do not depend strictly on the number of D1 and D2 primary ligand recognition sites.

Changes of amino acid and monoamine levels after neonatal 6-hydroxydopamine denervation in rat basal ganglia, substantia nigra, and Raphe nuclei.

The effects of a neonatal dopaminergic deafferentation with the neurotoxin 6-hydroxydopamine (6-OHDA) on endogenous tissue levels of catecholamines, indoleamines, and amino acids were investigated in discrete rat brain regions. After producing the lesion at postnatal day 3 by intraventricular injections of 6-OHDA, with a desipramine pretreatment to protect noradrenaline neurons, the animals were kept for 3 months. Their brains were dissected to obtain samples of neostriatum, Globus pallidus, Substantia nigra, and Raphe nuclei, which were then analyzed by high-performance liquid chromatography, coupled either to electrochemical detection for aromatic monoamines, or to post-column ninhydrin derivatization with spectrophotometry for amino acids. The neonatal 6-OHDA treatment depleted dopamine (DA) levels in neostriatum, Globus pallidus, and Substantia nigra, but in Raphe nuclei DA was increased. The main metabolites of DA were also decreased in neostriatum, Globus pallidus, and Substantia nigra but remained unchanged in Raphe nuclei. Serotonin (5-HT) and its metabolite 5-hydroxy-indole-3-acetic acid increased in neostriatum and Raphe nuclei; in Substantia nigra there was a slight increase in 5-HT only. The 6-OHDA lesion caused heterogeneous alterations in amino acid contents, which varied according to the region. In the neostriatum there were increases of gamma-aminobutyric acid (GABA), aspartic acid, and glycine. In the Globus pallidus taurine, GABA, glutamic acid, glutamine, aspartic acid, serine, and alanine were elevated. In the Substantia nigra only increases in taurine, GABA, glutamic acid, and glutamine could be documented. This study shows important changes in amino acid levels and in some of their ratios, occurring in different anatomical subdivisions of the basal ganglia and related brainstem nuclei following a neonatal treatment with 6-OHDA. The results thus demonstrate major biochemical modifications in amino acids in the aftermath of a DA denervation and/or a 5-HT hyperinnervation during an early developmental period.

[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.

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.

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

The amino acid gamma-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 phosphatidylethanolamine 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]raclopride binding to neostriatal dopamine D2 receptors: role of disulfide and sulfhydryl groups.

Receptor binding studies were performed in rabbit neostriatum (caudate-putamen) using the dopamine D2 antagonist [3H]raclopride. Treatment of the membrane preparations with the reducing agent L-dithiothreitol (L-DTT) as well as with the alkylating compound N-ethylmaleimide (NEM), produced dose-dependent decreases of specific [3H]raclopride binding; the IC50 values were of 3.1 and 1.2 mM, respectively. Saturation experiments showed that the reduction of disulfide (-S-S-) bonds by L-DTT (1 mM) decreased the number of binding sites, with only a slight increase in the affinity. On the other hand, alkylation of sulfhydryl (-SH) groups by NEM (1 mM) decreased both receptor number and affinity. The properties of the remaining binding sites were examined in competition curves with the physiological substrate dopamine and the dopaminergic antagonist (+)butaclamol. The IC50 values for (+)butaclamol in control and in L-DTT and NEM treated membranes were between 3.4 and 4.8 nM, with Hill coefficients (nH) of 1, indicating that the remaining binding sites conserved a high affinity for antagonist binding. In the case of dopamine, the curves were shallow (nH 0.45-0.64) and both compounds increased the IC50 from 0.7 microM (control) to 8 microM and 11 microM, for L-DTT and NEM respectively. Iterative analysis revealed that L-DTT produced a very important (greater than 60%) decrease in the number of high-affinity (RH) binding. After NEM, there was a decrease in both the number of (RH) and the affinity (KH) of the high-affinity binding sites, and in the affinity (KL) of the low-affinity sites. These results demonstrate the participation of -S-S- and -SH groups in the agonist conformation of the primary ligand recognition site of the dopamine D2 receptor. Alternatively, -S-S- and -SH groups could be related to the coupling of the primary ligand recognition protein with adenylate cyclase by means of an inhibitory type of G protein.

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 ionic 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.

[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.

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.

[3H]paroxetine binding and serotonin content of rat and rabbit cortical areas, hippocampus, neostriatum, ventral mesencephalic tegmentum, and midbrain raphe nuclei region.

The high-affinity binding of [3H]paroxetine to membranes was measured in different regions of the rat and rabbit brain: cingulate, frontal, parietal, piriform, entorhinal, and visual cortical areas; dorsal and ventral hippocampus; rostral and caudal halves of neostriatum (rat) or caudate nucleus and putamen (rabbit); ventral mesencephalic tegmentum; and midbrain raphe nuclei region. The tissue concentrations of serotonin (5-HT), 5-hydroxyindole-3-acetic acid (5-HIAA) and 5-hydroxy-l-tryptophan (5-HTP) were also determined by high-performance liquid chromatography (HPLC) in the same brain samples. The regional density of [3H]paroxetine binding varied in both species; the highest values (Bmax) were found in the midbrain raphe region and ventral mesencephalic tegmentum. The cortical values ranged from moderate to low, with a significantly higher density in the cingulate cortex of the rat compared with rabbit. In the rat, there was also a higher density in the ventral than dorsal hippocampus, and the caudal than rostral neostriatum. In the rabbit, the hippocampal and neostriatal values were generally lower and more uniform. In both species, there was an excellent correlation between regional 5-HT levels and specific [3H]paroxetine binding (r = 0.87 in the rat and 0.96 in the rabbit). Considering the available quantitative data on the number of 5-HT nerve cell bodies and axon terminals in different regions of the rat brain, it appears likely that the high amount of [3H]paroxetine binding in the midbrain raphe region and ventral mesencephalic tegmentum reflects the presence of 5-HT uptake sites on 5-HT nerve cell bodies and dendrites as well as axon terminals. In other brain regions, the heterogeneous distribution of [3H]paroxetine binding parallels that of the number of 5-HT axon terminals, emphasizing the potential usefulness of this radioligand as a marker of 5-HT innervation density.