Bridget Hulihan-Giblin

Identification of Tryptophan 2,3-Dioxygenase RNA in Rodent Brain

Abstract: The gene for tryptophan 2,3‐dioxygenase (TDO) heretofore was believed to be expressed only in liver. The data presented here demonstrate that RNA encoding TDO is present in rodent brain. Oligonucleotide primers based on the rat liver TDO cDNA sequence were synthesized and used to amplify RNA derived from mouse whole brain and liver and rat brain regions by the RNA‐PCR. Reaction products were purified and subjected to DNA sequencing. Identical sequences were obtained when mouse whole brain and liver RNAs were amplified, and these sequences were shown to be 96% identical to the published rat liver tryptophan TDO cDNA sequence. In addition, TDO sequences were found in RNA derived from rat brainstem, cerebellum, cortex, hypothalamus, and the remainder of the brain.

Agonist-Promoted Down-Regulation and Functional Desensitization in Two Naturally Occurring Variants of the Human Serotonin1A Receptor

We recently reported two naturally occurring polymorphisms of the human serotonin1A (5-HT1A) receptor: glycine22→serine (Ser22) and isoleucine28→valine (Val28) in the putative amino-terminal domain of the receptor. To investigate the regulatory properties of these variants, the wild type (WT) and variant 5-HT1A receptors were stably expressed in CHO-K1 cells. WT, Ser22, and Val28 displayed similar high-affinity binding to [3H]-8-OH-DPAT. Competition experiments with 5-HT1A agonists and antagonists demonstrated similar pharmacological profiles. Receptor agonist-promoted down-regulation was tested by exposure to 100 μmol/L 8-OH-DPAT. After 24-h exposure, WT and Val28 underwent 59.3 ± 3.9% and 59.5 ± 1.4% reduction in receptor density respectively, whereas the degree of down-regulation was significantly lower for Ser22 (21.4 ± 4.2%). Cell treatment for 24 h with 100 μmol/L 8-OH-DPAT reduced the 5-HT-induced inhibition of cAMP accumulation by 24.9 ± 5.1% for WT and 16.4 ± 0.8% for Val28, but only by 4.8 ± 3% for Ser22. We conclude that the Ser22 variant is capable of attenuating agonist-mediated receptor down-regulation and desensitization.

Characteristics of [3H](+)-amphetamine binding sites in the rat central nervous system

Recent studies in our laboratory have demonstrated the presence of specific binding sites for [3H] (+)-amphetamine in crude membrane preparations derived from rat brain. In this report we have further characterized the specific binding of [3H] (+)-amphetamine in various subcellular fractions of rat brain and demonstrate a greater than five-fold enrichment in the crude synaptosomal (P2) fraction compared to a crude membrane preparation. Specific [3H] (+)-amphetamine binding in crude synaptosomal membranes is saturable and stereospecific with an apparent dissociation constant, Kd, of 2.8 +/- 0.5 microM and an estimated maximum number of binding sites, Bmax, of 60.4 +/- 8.4 pmoles/mg protein derived by Scatchard or Klotz analysis of binding data using filtration assays. Centrifugation assays yield a similar Kd though the apparent Bmax is higher. In addition specific [3H] (+)-amphetamine binding is: rapidly reversible, temperature sensitive, labile to preincubation in Tris buffer, inhibited by sodium ions and unevenly distributed in various brain regions. Specific [3H] (+)-amphetamine binding sites are found almost exclusively in the rat central nervous system (the brainstem, hypothalamus, and striatum exhibiting relatively high levels of binding), whereas peripheral tissues such as liver, kidney and heart have very low to undetectable levels of specific binding.

Analysis of the 5-HT1C receptor and the serotonin uptake site in fawn-hooded rat brain.

Both the 5-HT1C receptor and the 5-HT uptake binding sites were measured in Fawn-Hooded, Sprague-Dawley and Wistar rats. Five brain regions were examined: frontal cortex, hippocampus, striatum, hypothalamus, and brainstem. We found significant differences in the Bmax and Kd values in various brain regions comparing Fawn-Hooded rats, with Sprague-Dawley and Wistar animals. The regional differences in receptor number and affinity in both the 5-HT1C receptor and the 5-HT uptake site in the Fawn-Hooded strain, relative to Wistar and Sprague-Dawley animals, provide support for the use of the Fawn-Hooded rat in serotonin dysfunction studies.

Differential effects of chronic antidepressant treatment on 5-HT1C receptor binding sites in Wistar rat brain.

The effects of chronic clomipramine, imipramine and clorgyline on 5-HT1C receptors were studied in discrete brain regions, in male Wistar rats, using [3H]mesulergine to label the receptor binding sites. Clorgyline treatment significantly reduced [3H]mesulergine binding (Bmax values) in both the hypothalamus and striatum compared to saline-treated animals. There were no differences in the maximum number of [3H]mesulergine binding sites following clorgyline in the hippocampus, frontal cortex or brainstem. Neither clomipramine or imipramine treatment resulted in any significant changes in 5-HT1C receptor number in the brain regions examined here. Furthermore, the Kd values (receptor affinity) for [3H]mesulergine binding were not significantly different comparing treatment groups to control animals. The significant changes in discrete brain regions following chlorgyline treatment suggest that 5-HT1C receptors may be involved in the clinical efficacy for the treatment of depression and other neuropsychiatric disorders.

Regional analysis of 5-HT1A receptors in two species of Peromyscus

Two species of deer-mice, Peromyscus maniculatus (P. man) and Peromyscus leucopus (P. leu), were compared for differences in 5-hydroxytryptamine1A (5-HT1A) receptor number and affinity. Both species enter into torpor; however, P. man enters spontaneous torpor with a higher frequency and for a longer duration than P. leu. Further, compared to P. leu a higher percentage of P. man exhibit daily torpor. Deer mice can be induced to enter torpor by a reduction in food supply, shortened photoperiods, and decreasing ambient temperature. Under these conditions, P. man enters into torpor more frequently, for longer durations, and with a higher percentage of individuals as compared to P. leu. [3H]8-OH-DPAT was used to label 5-HT1A brain receptors in three brain regions: the frontal cortex, brainstem, and striatum. In addition, the hypothalamus and hippocampus were examined for 5-HT1A receptor differences; however, no measurable specific binding could be determined in these regions. In the frontal cortex, the Bmax values were significantly lower in P. man compared to P. leu. There were no significant differences in the Bmax values in the striatum and brainstem between P. man and P. leu. Further, there were no significant differences in the Kd values between the two species in any of the brain regions examined. The absence of any difference in receptor number or affinity in any of the brain regions examined, except the cortex, suggests that the 5-HT1A receptor is most likely not involved in a more efficient pathway to torpor.

Diurnal rhythms of 5-HT1A and 5-HT2 receptor binding in euthermic and torpor prone deermice, Peromyscus maniculatus

Deermice display both spontaneous and induced daily torpor bouts, attaining minimum body temperatures of 15-20 degrees C. There is evidence that brain serotonin may be involved in the initiation and/or maintenance of torpor. Inhibition of serotonin [5-hydroxytryptamine (5-HT)] synthesis markedly reduces the duration and depth of torpor. Because a certain percentage of deermice will not enter torpor under any circumstances, we were able to compare 5-HT receptor subtypes in deermice that readily enter into torpor (TP) and in non-torpor prone (NTP) animals. Deermice were trapped in the wild and subjected to food rationing and low ambient temperature and then sacrificed either in a normothermic or torpid state at 11:00 p.m. or 11:00 a.m. Whole brain was assayed for 5-HT1A and 5-HT2 receptor differences using [3H]8-OH-DPAT and [3H]ketanserin, respectively. The Bmax values for 5-HT1A receptors were significantly greater in both TP and NTP animals sacrificed at 11:00 p.m. compared to animals sacrificed at 11:00 a.m. In contrast, the density of 5-HT2 receptors was significantly greater in animals sacrificed at 11:00 a.m. compared to animals sacrificed at 11:00 p.m. This is consistent with the opposing functions of these receptors in the regulation of temperature and sleep. The affinity (Kd) of each receptor was unchanged. A comparison of TP and NTP animals sacrificed at the same time of day revealed no significant differences in either Bmax or in Kd values, indicating that differences in 5-HT1A and 5-HT2 receptors may not explain the heterogeneity of deermice in their ability to enter torpor.(ABSTRACT TRUNCATED AT 250 WORDS)

Brain Recognition Sites for Methylphenidate and the Amphetamines

The stimulants methylphenidate, cocaine, and amphetamine remain among the most widely abused psychotropic drugs. However, amphetamine has long been considered a prototypic anorectic agent and, in the past, was used in the treatment of obesity. Although amphetamine has been used as a therapeutic agent, amphetamine can be a potent psychotomimetic, and tolerance can develop to its anorectic effects. Consequently, the medical use of amphetamine has now been restricted to narcolepsy and childhood hyperkinesis. Methylphenidate is currently used in the treatment of attention-deficit disorder and minimal brain dysfunction in children to increase attention span and reduce hyperactivity. Methylphenidate also has been proposed as an antidepressant and can potentiate conventional antidepressant medications in patients suffering from atypical depression. Nevertheless, there is widespread illicit use of psychostimulants, in particular, because of the euphoriant and fatigue-reducing effects of methylphenidate, cocaine, and various amphetamine compounds.