Karley Y. Little

Regulation of Serotonin1A, Glucocorticoid, and Mineralocorticoid Receptor in Rat and Human Hippocampus: Implications for the Neurobiology of Depression

BACKGROUND Disturbances of the limbic-hypothalamic-pituitary-adrenal axis and the serotonin system are commonly found in depressive illness. Studying the effect of stress on these two neurobiological systems may give us important clues into the pathophysiology of affective illness and help us understand how stress and mood disorders are related. METHODS We studied the effect of chronic unpredictable stress and antidepressant treatment on serotonin 1A (5-HT1A), glucocorticoid (GR), anti mineralocorticoid (MR) receptor levels in rat hippocampus, using in situ hybridization and receptor autoradiography. We also used in situ hybridization to quantify hippocampal 5-HT1A, GR, and MR messenger (mRNA) levels in a small group of suicide victims with a history of depression, compared to matched controls (n = 6). RESULTS We found that rats subjected to chronic unpredictable stress showed a significant elevation of basal plasma corticosterone compared to nonstressed rats. Chronic stress also caused a decrease in 5-HT1A mRNA and binding in the hippocampus. In addition, chronic stress produced alterations on the MR/GR mRNA ratio in this same region. The decreases in 5-HT1A mRNA and binding, as well as the MR/GR alterations, were prevented in animals that received imipramine or desipramine antidepressant treatment. Zimelidine was unable to reverse stress-induced increases in corticosterone, and was only partially successful in preventing the stress-induced receptor changes in the hippocampus. Suicide victims with a history of depression showed changes that were very similar to the changes found in chronic stress. CONCLUSIONS Alterations in hippocampal 5-HT1A levels and in the MR/GR balance may be one of the mechanisms by which stress may trigger and/or maintain depressive episodes.

Dopamine Receptor Gene Expression in the Human Medial Temporal Lobe

The distributions of the messenger RNA molecules encoding the five known dopamine receptors have been determined in the medial temporal lobe of postmortem human brain. All five receptor mRNAs are present in temporal lobe structures, although their distributions are heterogeneous. The D1-like receptors, D1 and D5, have strikingly dissimilar distributions. D1 receptor mRNA is abundant in temporal neocortex but is rare elsewhere. D5 receptor message, however, is seen in the hippocampus, subicular complex, and in temporal cortex. The D2-like receptors have similar distributions: D2, D3, and D4 receptor mRNAs are all identifiable in the hippocampal formation and in the cortical regions of the medial temporal lobe. Distinct patterns of relative regional concentrations for each message are observed, however, suggesting a neuroanatomical substrate for potential differences in dopaminergic regulation within discrete regions of the medial temporal lobe. These results provide a description of the distribution of these receptor mRNAs in normal humans and suggest multiple levels of complexity as well as regulation of the medial temporal lobe dopamine projection.

Cocaine use increases [3H]WIN 35428 binding sites in human striatum

Animal studies suggest that chronic cocaine exposure may increase the function and/or synthesis of the dopamine transporter (DAT) under certain conditions, but the literature is complex. In order to test the hypothesis that cocaine exposure alters the DAT in humans, preliminary studies were done characterizing [3H]WIN 35428 binding in human striatum from normal controls. Following these experiments, the effects of chronic cocaine were examined in post mortem striatal specimens from 7 cocaine users and 7 controls matched for age and post mortem interval, employing quantitative autoradiography. Initial saturation experiments indicated that a one-site model was preferred with a Kd of 11 +/- 4 nM. [3H]WIN 35420 binding was then examined in cocaine users and controls at 0.5, 5, 10, and 50 nM radioligand concentrations. At each concentration of [3H]WIN 35420, optical densities for cocaine-exposed subjects were increased in caudate, putamen, and accumbens. The results suggest that total numbers of binding sites were increased in cocaine users. Based on the present and previous results, it appears that the regulation of the DAT is fairly plastic, and is highly sensitive to cocaine dosing regimes and withdrawal intervals. Chronic adaptations induced by cocaine in the DAT could contribute to the symptoms of binging, withdrawal depression, and/or craving.

Serotonin transporter binding sites and mRNA levels in depressed persons committing suicide

The serotonin transporter (5-HTT) has been found altered in postmortem brain samples from persons committing suicide, but the results of radioligand binding studies have been inconsistent. In the present series of experiments, autoradiographic radioligand binding and in situ hybridization techniques were utilized to examine 5-HTT function in the brains of 8 depressed subjects who had committed suicide, and matched controls. It was hypothesized that depressed subjects would demonstrate decreased numbers of 5-HTT binding sites and mRNA; however, [125I]RTI-55 binding to the 5-HTT was not different in the midbrain, hippocampus, or frontal cortex of depressed subjects. Also, 5-HTT mRNA levels in dorsal and median raphe nuclei were not different between controls and depressed subjects. The current results, although limited in scope because of the small number of subjects included, offer no evidence that alterations in the 5-HTT occur in pertinent brain regions of depressed individuals.

[125I]RTI-55 Binding to Cocaine-Sensitive Dopaminergic and Serotonergic Uptake Sites in the Human Brain

[125I]RTI‐55 is a newly synthesized cocaine congener that may offer advantages over other ligands previously used to examine cocaine binding sites. However, the in vitro pharmacological and anatomical characterization of [125I]RTI‐55 binding sites has not been previously performed in human brain. To determine the specificity, stability, and feasibility of [125I]RTI‐55 for use in radioligand binding assays in postmortem human tissue, a series of experiments were performed characterizing [125I]RTI‐55 binding sites in human brain using homogenized membrane preparations and quantitative autoradtography. Analysis of the association, dissociation, and saturation data favored two‐phase processes. A curve‐fitting analysis of the data derived in saturation experiments found a high‐affinity site with KD= 66 ± 35 pM and Smax= 13.2 ± 10.1 pmol/g of tissue and a low‐affinity site with KD= 1.52 ± 0.55 nM and Bmax of 47.5 ± 11‐2 pmol/g of tissue. Competition by ligands known to bind to the dopamine transporter showed a rank order of RTI‐55 > GBR‐12909 > mazindol > WIN 35428 > = methylphenidate > (−)‐cocaine > buproprion > (±)‐amphetamine. Binding to serotonergic sites was evaluated in the midbrain. Results of the saturation experiment performed autoradiographically in the midbrain showed a single site with KD= 370 ± 84 pM. It appears that [125I]RTI‐55 should be useful in further studies of the regulation of cocaine binding sites using postmortem human specimens.

Autoradiographic characterization of [3H]imipramine and [3H]citalopram binding in rat and human brain: species differences and relationships to serotonin innervation patterns

The neuroanatomical distribution of binding sites for [3H]imipramine and [3H]citalopram was assessed by in vitro autoradiography in select regions of the rat and human forebrain. To determine involvement of serotonin-containing terminals in the binding of [3H]imipramine and [3H]citalopram, binding of these compounds was measured in rats after destroying serotonin-containing neurons with 5,7-dihydroxytryptamine (5,7-DHT). Treatment with this neurotoxin decreased serotonin content by 90% and reduced [3H]citalopram binding to a similar extent. These results demonstrate that [3H]citalopram binding is a reliable marker for serotonin-containing terminals. Binding of [3H]imipramine was reduced by only 15-35% after 5,7-DHT treatment. These latter results suggest that only a small fraction of [3H]imipramine binding to brain sections is associated with serotonergic terminals under standard conditions used in autoradiographic studies with the ligand. Dose-response effects of fluoxetine and desipramine on displacement of [3H]imipramine binding in forebrain regions indicate that the ligand labels predominantly high capacity, low affinity binding sites. To determine the utility of the rat brain as a model for [3H]imipramine and [3H]citalopram binding in the human brain, binding of the ligands was compared in human and rat hypothalamus, amygdala, and hippocampus. The pharmacological characteristics of [3H]imipramine and [3H]citalopram binding were similar in the rat and human brain. However, substantial species differences were observed in topographic patterns of [3H]imipramine binding within the hippocampus and hypothalamus. The distribution of [3H]citalopram binding sites within the amygdala and hypothalamus were also strikingly different in rats compared to humans. This work provides the first demonstration that marked species differences exist in the topography of serotonergic innervation and in the distribution of [3H]imipramine binding sites within the rat and human brain regions examined.

Effects of cocaine on dopamine receptor gene expression: A study in the postmortem human brain

The effects of chronic cocaine exposure on dopamine D1 and D2 receptor gene expression in the human brain were studied in postmortem samples from chronic cocaine abusing and matched control subjects. Using in situ hybridization of receptor autoradiography to examine messenger ribonucleic acid (RNA) and binding sites, respectively, neither D1 nor D2 receptor expression was found to be changed in the nucleus accumbens, caudate, putamen, or substantia nigra of the cocaine-exposed subjects. Although chronic cocaine exposure can produce alterations in dopaminergic neurotransmission, sustained compensatory changes in dopamine receptor expression do not appear to occur in the human.

β-adrenergic receptor binding in frontal cortex from suicide victims

Beta-adrenergic binding in frontal cortex samples from suicide victims has been reported to be increased, unaltered, and decreased compared to matched controls. Subjects diagnoses and drug exposures in these studies were not equivalently documented and were possibly different. In the present study, diagnostic and symptomatic information was systematically collected from family members of 15 subjects committing suicide and 15 matched controls using standardized interview techniques. The goal was to test the hypothesis that alterations in beta-adrenergic binding were more likely to be found in subjects with evidence of depressive disorders. [125I]pindolol binding in frontal cortex was found to be significantly lower in the group committing suicide compared to the matched controls (21.1 +/- 1.1 fmol/mg protein vs. 24.8 +/- 0.8 fmol/mg protein, p < .02). However, no diagnostic subgroup among the suicide victims appeared distinct.

Species differences in regional patterns of 3H-8-OH-DPAT and 3H-zolpidem binding in the rat and human brain.

The rat has proven to be a valuable preclinical model for characterizing effects of psychotrophic drugs and for identifying new psychotherapeutic agents in pharmacological screens. However, substantial differences have been described between the rat and human brain in regard to the neuroanatomical distribution of some drug and neurotransmitter receptor binding sites. To assess the utility of the rat as a model for the neuroanatomical topography of 5-HT1A and type 1 benzodiazepine (BDZ) receptors in humans, the distribution of binding sites for 3H-8-OH-DPAT (5-HT1A agonist) and 3H-zolpidem (type 1 BDZ agonist) was compared with autoradiography in select regions of the rat and human brain. Concordance in the binding patterns for the two ligands was observed in several brain regions for the two species. However, substantial differences were also found in the topography of binding sites for the ligands in the rat and human brain. High 3H-8-OH-DPAT binding was seen in the dorsal raphe nucleus and hippocampal formation in both the rat and human brain. However, species differences were observed in the relative distribution of ligand binding among hippocampal subregions. In the cerebral cortex, the laminar distribution of 3H-8-OH-DPAT binding sites was notably different for rats and humans. In humans, outer cortical layers were most densely labeled with 3H-8-OH-DPAT, whereas in the rat cortex, the highest binding was in the inner layers. A striking difference between rats and humans was observed for 3H-8-OH-DPAT binding in the lateral septal nucleus, which was densely labeled in the rat but weakly labeled in humans. Substantial differences between rats and humans were also observed for 3H-zolpidem binding. In the rat brain, high densities of binding sites were found in the medial septum, inferior colliculus, and substantia nigra reticulata. These regions showed very low 3H-zolpidem binding in the human brain. Intermediate binding was seen in the rat cerebral cortex, and low binding was found in the hippocampus. By contrast, in humans, cerebral cortical regions were the most densely labeled of all regions studied, and certain hippocampal subregions exhibited relatively high binding. The striking neuroanatomical differences in 3H-8-OH-DPAT and 3H-zolpidem binding observed between rats and humans suggest that different functional consequences may be produced within specific brain regions after administration of drugs that influence 5-HT1A and type 1 BZD receptors.

Effect of Cocaine on Dopamine Transporter Receptors Depends on Routes of Chronic Cocaine Administration

Investigation of residual behavioral states produced by withdrawal from different routes of cocaine administration indicates that depending on the mode of intake, chronic cocaine produces either tolerance or sensitization to subsequent challenge doses of cocaine. We studied the effect of routes of cocaine administration on the dopamine transporter receptors (DATR), the presumed neuronal mediator of cocaine reward, using the diphenyl substituted piperazine derivative, [3H]GBR 12935 and the cocaine analogue [3H]WIN 35,428. Alzet osmotic mini-pumps filled with either cocaine (100 mg/ml) or saline were surgically implanted on rats into a subcutaneous pocket at the dorsal midline, continuously infusing cocaine at the rate of 40 mg/kg/day. The pumps were removed 14 days later, and the rats were killed 7 days after the removal of pumps. For the intermittent administration, two groups of rats were injected daily either with 40 mg/kg of cocaine or saline for 14 days. Rats were killed 7 days following the last injection. Continuous infusion of cocaine did not alter the [3H]GBR 12935 dopamine transporter (DAT) binding either in the caudate nucleus or in the prefrontal cortex, but it enhanced the density of [3H]WIN 35,428-labeled DAT receptor sites in the caudate putamen. In contrast, intermittent administration of cocaine resulted in a selective alteration of [3H]GBR 12935 binding in the prefrontal cortex but not in the caudate; the cocaine-injected rats had a 48% decrease in [3H]GBR 12935, Bmax (p < .05), without changing the KD. The contrast between the lack of effect of cocaine on [3H]GBR 12935-DATR and the increased binding of [3H]WIN 35,428-DATR highlights the differential sensitivities of the two binding sites to the continuous presence of cocaine.